Use of penetration enhancers and barrier disruption methods to enhance the immune response of antigen and adjuvant

ABSTRACT

A transcutaneous immunization system where the topical application of an adjuvant and an antigen or nucleic acid encoding for an antigen, to intact skin induces a systemic or mucosol antibody response. The immune response so elicited can be enhanced by physical or chemical skin penetration enhancement.

RELATED APPLICATIONS

The present application is a continuation application of U.S.application Ser. No. 09/257,188, filed Feb. 25, 1999, now U.S. Pat. No.6,797,276, which is a continuation-in-part of U.S. application Ser. No.08/749,164, filed Nov. 14, 1996, now U.S. Pat. No. 5,910,306; U.S.application Ser. No. 08/896,085, filed Jul. 17, 1997, now U.S. Pat. No.5,980,898; and PCT/US97/21324 designating the U.S., filed Nov. 14, 1997,now abandoned, published under PCT Article 21(2) in English. U.S.application Ser. No. 09/257,188 claims the benefit of U.S. ProvisionalApplication No. 60/075,850, filed Feb. 25, 1998; U.S. ProvisionalApplication No. 60/075,856, filed Feb. 25, 1998; and U.S. ProvisionalApplication No. 60/086,251, filed May 21, 1998. All of the foregoingdocuments are herein incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to transcutaneous immunization using anADP-ribosylating exotoxin or other adjuvants with an antigen, and theuse of penetration enhancers and barrier disruption agents to enhancethe immune response. The invention also relates to activation of theantigen, adjuvant, their targets in the skin, or a combination thereofto enhance the antigen-specific immune response induced thereto.

2. Description of the Related Art

Skin, the largest organ of the human body, is an important part of thebody's defense against invasion by infectious agents and contact withnoxious chemicals (see Bos, 1997). Unwanted skin reactions such asallergic or atopic dermatitis are known, but induction of a systemicimmune response by application of an adjuvant and antigen which elicitsspecific immune effectors and provides a therapeutic advantage by simpleapplication of adjuvant and antigen to skin does not appear to have beentaught or suggested prior to our invention.

Cholera toxin (CT) and heat labile interotoxin from E. coli (LT) areexamples of a noxious chemical, which one would have expected theprotective layers of skin to protect against penetration by the noxiussubstances. Craig (1965) reported that stool filtrates of cholerapatients injected intracutaneously into rabbits or guinea pigs produceda characteristic delayed, sustained edematous induration (swelling),which was induced by the presence of toxin in the skin. The swelling andvascular leakage was so dramatic that it was ascribed to an unknownpermeability factor which was later shown to be CT itself. Thus, onecould have reasonably expected that CT would be extremely reactogenicwhen placed on the skin, if it were to enter the skin, causing similarredness and swelling. The Craig test injecting CT into the skin, becamea standard measurement for the presence and amount of CT in stoolfiltrates or culture media. Data confirmed that this skin reactivity wasdue to cholera toxin (see Finkelstein and LoSpallutto, 1969).

Craig (1965) cautioned, “The absence of skin lesions in clinical choleracertainly does not preclude the possibility that the noxa responsiblefor gut damage could also have a deleterious effect upon the skinprovided it is applied to skin in sufficient concentration”. The extremereactogenicity of cholera toxin in the skin was used as a test for itstoxicity and the prior art evidenced an expectation that cholera toxinwould be reactogenic if applied to the skin, producing an undesirablereaction. Such adverse reactions have been well documented by knownauthorities in the field (Craig, 1972).

In contrast, we have shown cholera toxin to be immunogenic, acting asboth antigen and adjuvant, when placed on the skin but without anyresulting local or systemic side effects. This lack of reactogenicitywhen cholera toxin was placed on the skin for transcutaneousimmunization was surprising and contradicted conclusions one would havedrawn from the prior art. Specifically, CT placed on the skin accordingto our invention acts as a non-toxic, non-reactogenic adjuvant, incontrast to the expectations of Craig, while injection of CT into theskin results in swelling and redness. Thus, it was not obvious prior toour invention that cholera toxin or other ADP-ribosylating exotoxins orallow adjuvants applied topically would be useful for transcutaneousimmunization. In fact large doses of heat labile enterotoxin (LT) placedon the skin of humans has been shown to induce a systemic immuneresponse without local or systemic toxicity.

This unexpected absence of reactogenicity is extremely important to theuse of vaccines. Vaccine antigens and adjuvants are useful whenimmunization produces a protective immune response without significantunwanted reactions. Historically, reactogenicity of vaccines such asswelling, tenderness and pain at the site of injection has in some cases(e.g., typhoid and pertussis) been accepted because of the benefits ofvaccination. However, high levels of reactogenicity and other sideeffects are not desirable, and would be problematic for development ofnew vaccine adjuvant and antigen candidates. Research efforts arefocused on making vaccine adjuvants that are stimulatory and do notinducing unwanted reactions. Whole cell pertussis vaccines inducesystemic and local side effects and, as a result, this effective vaccineand time tested vaccine is being replaced by acellular pertussisvaccines solely because they are less reactogenic.

The present invention differs from that of U.S. Pat. No. 5,830,877 whichteaches the use of a naked plasmid that encodes for biologically activepeptides into a mammalian host. The invention described herein teachesthe use of an adjuvant and antigen or nucleic acid administered togetheron the skin to induce an immune response. The invention described hereinfurther differs from that of U.S. Pat. No. 5,830,877 which teaches awayfrom the use of peptides that are not encoded in a nucleic acid andproduced by the host cell because of the toxicity associated withbiologically active peptides, the problems and cost of isolating,purifying and synthesizing peptides and their short half life in vivoresulting form degradation by proteases present in the target tissue.This clearly teaches away from the addition of an adjuvant such acholera toxin to a coadministered antigen or nucleic acid. In fact thenovelty of the ability of a large molecule such as CT to induce animmune response by application through the skin without toxicity has ledto a number of scientific papers describing this novelty and publicexcitement over the potential implication of delivery of proteins forvaccination by skin application. Unlike U.S. Pat. No. 5,830,877 thepresent invention is not dependent on stimulation of local inflammationor irritation. Unlike U.S. Pat. No. 5,830,877 the invention does notdepend on irritation or local inflammation to increase the permeabilityof cell membranes to enhance the uptake of the antigens, plasmids orRNA. In fact the striking feature regarding Transcutaneous Immunizationis the absence of local inflammation.

Unlike the present invention, U.S. Pat. No. 5,824,313 teaches theapplication of extremely small (less than 500 daltons) lymphoid organmodifying agents such as 1,25-dihydroxy-16-ene Vitamin D.sub.3 andcalcipotriene or dehydroepiandrosterone (DHEA), DHEA congeners andDHEA-derivatives with the intramuscular injection of an antigen toaffect antibody responses.

Transcutaneous immunization requires both passage of an antigen throughthe outer barriers of the skin, which was thought to be impervious tosuch passage, and an immune response to the antigen. Fisher's ContactDermatitis states that molecules of greater than 500 daltons cannotnormally penetrate the skin. There is a report by Paul et al. (1995) ofinduction of an immune response with transferosomes, a lipid structuredistinct from liposomes. In this publication, the transferosomes wereused as a vehicle for antigen (bovine serum albumin and gap junctionproteins) and complement-mediated lysis of antigen-sensitized liposomeswas assayed. The limit to penetration of the skin by antigen was statedto be 750 daltons. In their study, an immune response was not inducedwhen a solution containing antigen was placed on the skin; onlytransferosomes were able to induce an immune response. Paul and Cvec(1995) also stated that it is “impossible to immunize epicutaneouslywith simple peptide or protein solutions”.

Such references explain why our successful use of a molecule likecholera toxin (which is 85,000 daltons) as an antigen or adjuvant inimmunization was greeted with surprise by the field because such largemolecules were not expected to penetrate the skin and, therefore, wouldnot be expected to induce a specific immune response.

However, we have shown in U.S. application Ser. No. 08/749,164 (filedNov. 14, 1996); U.S. application Ser. No. 08/896,085 (filed Jul. 17,1997); and international application PCT/US97/21324 (filed Nov. 14,1997) that using an ADP-ribosylating exotoxin, such as cholera toxin, asan antigen could elicit a strong antibody response that is highlyreproducible. When an ADP-ribosylating exotoxin, such as cholera toxin,was used as an immunoadjuvant and applied to the skin in a salinesolution with a separate antigen (e.g., diphtheria toxoid), a systemicand mucosal antigen-specific antibody response could be elicited. In thepresent application, we disclose that transcutaneous immunization usinga penetration enhancer, a barrier disruption agent, or combinationsthereof may improve the adjuvant activity of a bacterial exotoxin.

We have shown that like cholera toxin (CT), heat-labile enterotoxin fromE. coli (LT), Pseudomonas exotoxin A (ETA), pertussis toxin (PT) and awide variety of antigens including killed rabies virus, recombinantssuch as HIV p55 gag, polysaccheride conjugates such as Hib, sonicates,pertactin for example, are able to pass through the skin and induce animmune response. Additionally CT, LT, ETA and PT and bacterial DNA andcytobines, can act as adjuvants to induce an immune response to antigensco-administered on the skin. Thus tetanus toxoid, not immunogenic byitself on the skin, can induce a strong immune response when placed onthe skin with CT. We have proposed that the Langerhans cell populationunderlying the site of application are a preferred antigen presentingcell for delivering antigen to the immune system. Adjuvant may act onthe antigen presenting cell directly, or through lymphocytes recognizingantigen.

We propose to enhance the immune response to transcutaneously adjuvantand/or antigen utilizing penetration enhancement techniques. Accordingto Hurley, “Skin owes its durability to the dermis, but its chemicalimpermeability resides in the epidermis and almost exclusively in itsdead outer layer, the stratum corneum”. For transcutaneous immunizationusing, for example, an ADP-ribosylating exotoxin as adjuvant and asoluble protein antigen such as diphtheria toxoid, penetration of thestratum corneum must occur. Penetration enhancement techniques would bedesigned to increase the movement of transcutaneous antigens andadjuvants through the stratum corneum layer of skin.

Further, we propose that transcutaneous immunization using activation ofat least one of the antigen, adjuvant and skin component will enhancethe immune response as assayed by quantitative and qualitativeparameters. Antigen-adjuvant of the formulation may be activated bytrypsin cleavage of a bacterial exotoxin (e.g., trypsin-cleaved LT, withor without reduction). Activation of the skin at the application site ofthe formulation may be accomplished by the use of barrier disruptionagents (e.g., acetone, alcohol) which increases the size or activationof the underlying Langerhans cell population, or by an enzyme orcombination of enzymes (e.g., enzymes with sialidase activity) whichincreases the amount or accessibility of ganglioside GM1 receptor.

SUMMARY OF THE INVENTION

An object of the invention is to provide an enhanced system fortranscutaneous immunization which induces an immune response (e.g.,humoral and/or cellular effector) in a subject, a subject being ananimal or human. This delivery system provides simple application tointact skin of an organism of a formulation comprised of antigen andadjuvant to induce a specific immune response against the antigen.Although not required for induction of an immune response by this simpledelivery system, supplementation of the aforementioned process withpenetration enhancement or barrier disruption may enhance immunizationand/or vaccination.

In particular, the adjuvant or antigen or skin may assist in thepenetration of the stratum corneum or epidermis to encounter the antigenpresenting cells of the immune system (e.g., Langerhans cells in theepidermis, dermal dendritic cells, dendritic cells, follicular dendriticcells, macrophages, B lymphocytes) and/or induce the antigen presentingcells to phagocytose the antigen. The antigen presenting cells thenpresent the antigen to T and B cells. In the instance of Langerhanscells, the antigen presenting cells then may migrate from the skin tothe lymph nodes and present antigen to lymphocytes (e.g., B and/or Tcells), thereby inducing an antigen-specific immune response.

In addition activation of the antigen, adjuvant, skin, or anycombination thereof may be accomplished to supplement the immunizationprocess.

In addition to eliciting immune reactions leading to generation of anantigen-specific B lymphocyte and/or T lymphocyte, including a cytotoxicT lymphocyte (CTL), another object of the invention is to positivelyand/or negatively regulate components of the immune system by using thetranscutaneous immunization system to affect antigen-specific helper(Th1 and/or Th2) or delayed-type hypersensitivity (DTH) T-cell subsets.This can be exemplified by the differential behavior of CT and LT whichcan result in different T-helper responses or different levels ofprotection in in-vivo challenge molds using transcutaneous immunization.

DESCRIPTION OF THE DRAWINGS

FIGS. 1 a-f are photographs showing no inflammation at the site ofimmunization (A,B), Langerhans cell activation by LT in human skin atthe site of immunization (C,E) and the absence of Langerhans cellactivation in skin from the contralateral arm (D,F).

FIGS. 2 a-d are photographs showing normal Langerhans cell (A,B, 200 and400×) and Langerhan cell activation by Cholera Toxin in mouse skin (C,D.200×, 400×).

DESCRIPTION OF PREFERRED EMBODIMENTS

In one embodiment of the invention, a formulation containing antigen andadjuvant such as CT and DT, is applied to intact skin of an organismafter penetration enhancement of the skin, the antigen is presented toimmune cells, and an antigen-specific immune response is induced withoutperforating the skin. The formulation may include additional antigens ornucleic acids such that transcutaneous application of the formulationinduces an immune response to multiple antigens, or nucleic acidsencoding for antigens preferably from 2 to 20 but possibly up to 200. Insuch a case, the antigens may or may not be derived from the samesource, but the antigens will have different chemical structures so asto induce immune responses specific for the different antigens.Antigen-specific lymphocytes may participate in the immune response and,in the case of participation by B lymphocytes, antigen-specificantibodies may be part of the immune response.

In another embodiment of the invention, the invention is used to treatan organism. If the antigen is derived from a pathogen, the treatmentvaccinates the organism against infection by the pathogen or against itspathogenic effects such as those caused by toxin secretion. Aformulation that includes a tumor antigen may provide a cancertreatment; a formulation that includes an allergen may be used to treatfor allergic disease; a formulation that includes an autoantigen mayprovide a treatment for a disease caused by the organism's own immunesystem (e.g., autoimmune disease). The invention may be usedtherapeutically to treat existing disease, protectively to preventdisease, or to reduce the severity and/or duration of disease.

In a further embodiment of the invention, a patch for use in the abovemethods is provided. The patch may comprise a dressing, and effectiveamounts of antigen or nucleic acids and adjuvant. The dressing may beocclusive or non-occlusive. The patch may contain penetration enhancersor may include a device for physical penetration enhancement. The patchmay include additional antigens such that application of the patchinduces an immune response to multiple antigens. In such a case, theantigens may or may not be derived from the same source, but theantigens will have different chemical structures so as to induce animmune response specific for the different antigens. For effectivetreatment; multiple patches may be applied at frequent intervals orconstantly over a period of time.

Moreover, in yet another embodiment of the invention, the formulation isapplied to intact skin overlying more than one draining lymph node fieldusing either single or multiple applications or a separate patch foradjuvant or antigen/nucleic acid. The formulation may include additionalantigens such that application to intact skin induces an immune responseto multiple antigens. In such a case, the antigens may or may not bederived from the same source, but the antigens will have differentchemical structures so as to induce an immune response specific for thedifferent antigens.

The formulation may be applied to the skin to boost or prime the immuneresponse in conjunction with other routes of immunization. Thus, primingwith transcutaneous immunization with either single or multipleapplications may be followed with oral, nasal, or parenteral techniquesfor boosting immunization with the same or altered antigens. Theformulation may include additional antigens such that application tointact skin induces an immune response to multiple antigens. In such acase, the antigens may or may not be derived from the same source, butthe antigens will have different chemical structures so as to induce animmune response specific for the different antigens.

In addition to antigen and activated adjuvant, the formulation maycomprise a vehicle. For example, the formulation may comprise AQUAPHOR(an emulsion of petrolatum, mineral oil, mineral wax, wool wax,panthenol, bisabol, and glycerin), emulsions (e.g., aqueous creams),microemulsions, gels, oil-in-water emulsions (e.g., oily creams),anhydrous lipids and oil-in-water emulsions, anhydrous lipids andwater-in-oil emulsions, fats, waxes, oil, silicones, and humectants(e.g., glycerol).

The antigen may be derived from a pathogen that can infect the organism(e.g., bacterium, virus, fungus, or parasite), or a cell (e.g., tumorcell or normal cell) or allergen or biological warfare agent. Theantigen may be a tumor antigen or an autoantigen. Chemically, theantigen may be a carbohydrate, glycolipid, glycoprotein, lipid,lipoprotein, phospholipid, polypeptide, or fusion protein (recombinant)or chemical conjugate of the above. The molecular weight of the antigenmay be greater than 500 daltons, preferably greater than 800 daltons,and more preferably greater than 1000 daltons.

Antigen may be obtained by recombinant means, chemical synthesis, orpurification from a natural source. One advantage of transcutaneousimmunization may be that purification of an antigen is not necessarye.g. a whole organism may be sonicated and used for immunization. Thelevel of toxicity associated with injecting a product from such apreparation is often too toxic to be tolerated, such as LPS, which canbe fatal if injected, but is non-toxic on the skin. Preferred areproteinaceous antigen or conjugates with polysaccharide. Antigen may beat least partially purified in cell-free form. Alternatively, antigenmay be provided in the form of a live virus, an attenuated live virus,or an inactivated virus, sonicated or lysed whole bacterium, parasite ordetergent treated virus, or fraction thereof.

Inclusion of an adjuvant may allow potentiation or modulation of theimmune response. Moreover, selection of a suitable antigen or adjuvantmay allow preferential induction of a humoral or cellular immune prmucosal response, specific antibody isotypes (e.g., IgM, IgD, IgA1,IgA2, IgE, IgG1, IgG2, IgG3, and/or IgG4), and/or specific T-cellsubsets (e.g., CTL, Th1, Th2 and/or T_(DTH)). Optionally, antigen,adjuvant, may be provided in the formulation by means of a nucleic acid(e.g., DNA, RNA, cDNA, cRNA) encoding the antigen or adjuvant asappropriate with a antigen or adjuvant that has been added to thenucleic acid. This technique is called genetic immunization.

The term “antigen” as used in the invention, is meant to describe asubstance that induces a specific immune response when presented toimmune cells of an organism. An antigen may comprise a singleimmunogenic epitope, or a multiplicity of immunogenic epitopesrecognized by a B-cell receptor (i.e., antibody on the membrane of the Bcell) or a T-cell receptor. A molecule may be both an antigen and anadjuvant (e.g., cholera toxin) and, thus, the formulation may containonly one component. Antigen may be provided as a whole organism such as,for example, a bacterium or virion; antigen may be obtained from anextract or lysate, either from whole cells or membrane alone; or antigenmay be chemically synthesized for produced by recombinant means.

The term “adjuvant” as used in the invention, is meant to describe asubstance added to the formulation to assist in inducing an immuneresponse to the antigen.

The term “effective amount” as used in the invention, is meant todescribe that amount of antigen which induces an antigen-specific immuneresponse. Such induction of an immune response may provide a treatmentsuch as, for example, immunoprotection, desensitization,immunosuppression, modulation of autoimmune disease, potentiation ofcancer immunosurveillance, or therapeutic vaccination against anestablished infectious disease.

By the epidermis we mean the cells of the skin from the basal layer ofkertinoctyes and basal lamina up to and through the stratum corneum.

The definition of transdermal is generally held to be: Relating to,being, or supplying a medication in a form for absorption through theskin into the bloodstream (˜drug delivery) (˜nitroglycerin) (˜nicotinepatch). ²Frederick C. Mish et al., eds., Merriam-Webster's CollegiateDictionary, 10^(th) ed. (Springfield, Mass.: Merriam-Webster,Incorporated, 1997), 861.

The term draining lymph node field as used in the invention means ananatomic area over which the lymph collected is filtered through a setof defined set of lymph nodes (e.g., cervical, axillary, inguinal,epitrochelear, popliteal, those of the abdomen and thorax).

Skin penetration may be enhanced using techniques that increase skinhydration. According to Roberts and Walker (1993), “The state ofhydration of the stratum corneum (SC) is one of the most importantfactors in determining the rate of percutaneous absorption of a givensolute”. The state of hydration interacts with the principal ofdiffusion in determining the rate of absorption of a substance throughthe skin. Furthermore, Hurley stated:

-   -   “Absorption of substances through the stratum corneum is        believed to occur by diffusion in accordance with Fick's laws of        diffusion in which the rate of absorption of a chemical is        proportional to the concentration difference across the        membrane. Thus, a concentration gradient between the high        concentration of solute on the skin surface and its absence or        low concentration below the stratum corneum is the driving force        in this process. Transcorneal movement of absorption is        classically represented as ‘percellular’, that is, directly        through the cell walls of the compacted corneum and not        intercellularly. Intracellular protein filaments are described        as the pathways for polar (water soluble) compounds and the        medium between filaments serves as the route for nonpolar        (lipid-soluble) substances . . . Hydration increases the        permeability of the stratum corneum for most substances by a        number of cytophysical mechanisms that are not completely        clarified.”        Thus, while skin hydration is generally known to enhance skin        penetration, the mechanisms by which this occurs are not        entirely clear and, thus, not predictable prior to the present        invention and not thought to allow penetration of large        molecules. (7750 daltons).

The use of vehicles for increasing hydration is well known. Occlusivedressings, such as vapor-impenetrable plastic films (e.g.,polyvinylidine, polyethylene) enhance absorbtion principally throughincreased hydration of the stratum corneum, a result of swelling of thecorneocytes, and uptake of water into the intercellular corridors.Hydrocolloid patches may also be used to enhance skin penetration.Absorbtion of steroids can be increase over 100 fold using plasticocclusive film. Generally, greases, oils or impermeable plastic inducethe most hydration by occlusion. See, for example, Idson (1978);Hollingsbee (1995); and Mckenzie and Stoughton (1962). The use ofhydration or vehicle for hydration with an antigen and adjuvant were notknown prior to our invention as penetration. The skin was thought to belimited to small molecules even in the hydrated state.

Suitable agents which are known to enhance absorption of drugs throughskin are described in Sloan, Use of Solubility Parameters from RegularSolution Theory to Describe Partitioning-Driven Processes, Ch. 5,“Prodrugs: Topical and Ocular Drug Delivery” (Marcel Dekker, 1992), andat places elsewhere in the text.

It is expected that these techniques (and others which areconventionally used to facilitate drug delivery) may be adapted topreparation of nucleic acids for use in the methods of the invention bythose of ordinary skill in the art without undue experimentation.Specific examples illustrating this suitability are set forth below.

The state of the art in skin penetration enhancement is described inPharmaceutical Skin Penetration Enhancement Edited by Kenneth A. Waltersand Jonathan Hadgraft, Published by Marcel Dekker, Inc., New York, 1993.

Skin permeability and/or skin hydration may be expected by selecting anappropriate vehicle from different classes, such as humectant (e.g.,glycols, glycerols), powder, (e.g., clays, shake lotions), oil/water(O/W) emulsion (e.g., aqueous creams), water/oil emulsion (e.g., oilycreams), emulsifying base (e.g., anhydrous lipid and O/W emulsifiers),absorbtion base (e.g., anhydrous lipid and W/O emulsifiers), lipophilic(e.g., fats, waxes, oils, silicones), and occlusive dressing (e.g.,plastic wrap).

Other methods that disrupt the stratum corneum proteins to enhancepenetration in the present invention may be employed. Salicylic acid isa keratinolytic that may increase absorption. Urea acts both as akeratinolytic and hydrater of the skin, and may act as a penetrationenhancer. Phospholipase A2 and phosphatidylcholine dependentphospholipase C may be used as epidermal enzymes to enhance penetration.Other penetration enhancers may include ethanol, acetone, detergents,bases, nair©, propylene glycol, pyrriolidones, dimethylacetamide,dimethylformamide, dimethylsulfoxide, alkyl sulfoxide, phosphine oxide,surfactants and caprolactams such as azone. Other compounds that may beused for penetration enhancement include amines and amides, alkylN,N-distributed-amino acetates, decylmethylsulfoxide, pyrrolidones,pirotiodecane (HPE-101), benzlyalkonium, benzylalkonium chloridepolymers, silicone based polymers, fatty acids, cyclic ureas, terpenes,liposomes, and cyclodextrins. Penetration enhancers are well known inthe art, for example as described in Pharmaceutical PenetrationEnhancement, (Marcel Dekker, 1993) Other techniques that may be employedfor penetration include iontophoresis, ultrasound, electroporation, tapestripping, the use of gene guns or other propellant devices, tines suchas used for TB tine tests (as provided by Mono-Vacc system) ormicroneedles which penetrate the outer surface of the skin, or abrasiveswhich remove the outer layers of the skin and lipid extraction.

A device which may be used for disruption of the stratum corneum (whichis distributed in the U.S. by Connaught Laboratories, Inc. ofSwiftwater, Pa.) consists of a plastic container having a syringeplunger at one end and a tyne disk at the other. The tyne disk supportsa multiplicity of narrow diameter tynes of a length which will justscratch the outermost layer of epidermal cells but not penetrate theepidermis. Each of the tynes in the MONO-VACC kit is coated with oldtuberculin; in the present invention, each needle may be coated with apharmaceutical composition of antigen/nucleic acid and adjuvant. Use ofthe device with the present invention may not be according to themanufacturer's written instructions included with the device productbecause when used with the present invention does not penetrate theepidermis. Hereto, the device may be used for surface disruption todisrupt the outermost layes of the skin, the stratum corneum and upperepidermis, to enhance the transcutaneous immunization. Similar deviceswhich may also be used in this embodiment are those which are currentlyused to perform allergy tests.

Other approaches include barrier disruption. Inhibition of cholesterolsynthesis using systemically administered HMG CoA reductase inhibitorsand similar drugs interfere with barrier function and may allow enhancedpenetration of the formulation component.

It is also conceivable that the skin can be transformed to enhance thetranscutaneous immune response. CT and LT exert their effect via theganglioside GM1 binding by the B subunit. Ganglioside GM1 is aubiquitous cell membrane glycolipid found in all mammalian cells. In thegastrointestinal tract, when the pentameric CT B subunit binds to thecell surface, a hydrophilic pore is formed which allows the A subunit topenetrate across the lipid bilayer. The skin contains gangliosides at aconcentration of 30-35 nmol NeuAC/gm. Skin gangliosides are possibletargets for initiating transcutaneous immunization via mechanisms suchas Langerhans cells activation as described above.

One possible method to activate the skin for enhancing the effect oftranscutaneous immunization with ADP ribosylating exotoxins byincreasing the number of GM1 ganglioside molecules in skin cells. Thiscould be achieved by activation of receptor cells using sialidase toconvert gangliosides that do not bind the toxin into thesialidase-stable cholera toxin binding ganglioside GGnSLC (gangliosideGM1):

-   -   “It is interesting that the cholera vibrio is perhaps the        best-known source of sialidase (or neuraminidase, as it is often        called). Could this sialidase play a part in the natural history        of the disease by making more receptors available for the toxin?        If so, should any active immunizing agent the disease contain an        anti-neuriminidase element? Incubation of intestinal scrapings        with sialidase leads to a considerable increase in their ability        to bind the toxin, which is due not only to conversion of        sialidase-labile ganglioside to cholera toxin-binding        ganglioside, but also, apparently to the unmasking of otherwise        unapproachable ganglioside binding sites possibly by breaking        down glycoproteins. Pretreatment of dog intestine with sialidase        makes it produce more fluid in response to cholera toxin;        treatment of adrenal cells with sialidase increases their        responsiveness to cholera toxin; pretreatment of pigeon red        cells with sialidase increases the activation of the adenylate        cyclase in them by cholera toxin.        The biochemistry of cholera, In: Cholera: The American        Scientific Experience, 1947-1980, van Heyningen, W. E., and        Seal, J. R., Eds, Waterview Press, Boulder, 1983, page 263        (citations omitted).

The effect of treatment of the skin with sialidase may enhance thebinding of an ADP ribosylating exotixin such as CT to the immune cellstargeted by transcutaneous immunization. This represents a kind ofactivation of the skin for transcutaneous immunization. Additionally,neuraminidase may act as an epidermal enzyme concurrently enhancingpenetration.

The use of a penetration enhancer may be used in conjunction withactivation of the skin. Activation of the skin for transcutaneousimmunization may also be after treatments such as acetone or alcoholswabbing. It has been shown that skin barrier disruption using acetoneswabbing of the skin increased Langerhans cell density by 80% andincreased the reaction to contact allergens in vivo. If the density ofLangerhans cells is increased, then the potency of the immune responsemay be increased. Similar chemical disruption might be expected toincrease the number of Langerhans cells and result in activation of theskin component of transcutaneous immunization, by tape stripping, sodiumdodecyl sulfate, the use of alcohol swabbing, or a depilatory such ascalcium hydroxide. See Proksch and Brasch (1996, 1997) for use ofpenetration enhancers and barrier disruption in allergic contactdermatitis.

Penetration enhancement may be achieved by performance of simplemaneuvers such as alcohol swabbing immediately prior to immunization, byconcurrent use of penetration enhancement compounds or techniques or bytechniques such as acetone swabbing 24 hours prior to increase thenumber of Langerhans cells.

Processes for preparing a pharmaceutical formulation are well-known inthe art, whereby the antigen and adjuvant is combined with apharmaceutically acceptable carrier vehicle. Suitable vehicles and theirpreparation are described, for example, in Remington's PharmaceuticalSciences by E. W. Martin. Such formulations will contain an effectiveamount of the antigen and adjuvant together with a suitable amount ofvehicle in order to prepare pharmaceutically acceptable compositionssuitable for administration to a human or animal. The formulation may beapplied in the form of an cream, emulsion, gel, lotion, ointment, paste,solution, suspension, or other forms known in the art. In particular,formulations that enhance skin hydration, penetration, or both arepreferred. There may also be incorporated other pharmaceuticallyacceptable additives including, for example, diluents, excipients,binders, stabilizers, preservatives, and colorings.

Without being bound to any particular theory but only to provide anexplanation for our observations, it is presumed that the transcutaneousimmunization delivery system carries antigen to cells of the immunesystem where an immune response is induced. The antigen may pass throughthe normal protective outer layers of the skin (i.e., stratum corneum)and induce the immune response directly, or through an antigenpresenting cell (e.g., macrophage, tissue macrophage, Langerhans cell,dendritic cell, dermal dendritic cell, B lymphocyte, or Kupffer cell)that presents processed antigen to a T lymphocyte (see Stingl et al.,1989; Streilein and Grammer, 1989; Tew et al., 1997). Optionally, theantigen may pass through the stratum corneum via a hair follicle or askin organelle (e.g., sweat gland, oil gland).

Transcutaneous immunization with bacterial ADP-ribosylating exotoxins(bAREs) may target the epidermal Langerhans cell, known to be among themost efficient of the antigen presenting cells (APCs). We have foundthat bAREs activate Langerhans cells when applied epicutaneously to theskin in saline solution. Adjuvants such as activated LT may greatlyenhance Langerhans cell activation. The Langerhans cells direct specificimmune responses through phagocytosis of the antigens, and migration tothe lymph nodes where they act as APCs to present the antigen tolymphocytes, and thereby induce a potent antibody response. Although theskin is generally considered a barrier to invading organisms, theimperfection of this barrier is attested to by the numerous Langerhanscells distributed throughout the epidermis that are designed toorchestrate the immune response against organisms invading via the skin.According to Udey (1997):

-   -   “Langerhans cells are bone-marrow derived cells that are present        in all mammalian stratified squamous epithelia. They comprise        all of the accessory cell activity that is present in        uninflammed epidermis, an in the current paradigm are essential        for the initiation and propagation of immune responses directed        against epicutaneously applied antigens. Langerhans cells are        members of a family of potent accessory cells (‘dendritic        cells’) that are widely distributed, but infrequently        represented, in epithelia and solid organs as well as in        lymphoid tissue.    -   “It is now recognized that Langerhans cells (and presumably        other dendritic cells) have a life cycle with at least two        distinct stages. Langerhans cells that are located in epidermis        constitute a regular network of antigen-trapping ‘sentinel’        cells. Epidermal Langerhans cells can ingest particulates,        including microorganisms, and are efficient processors of        complex antigens. However, they express only low levels of MHC        class I and II antigens and costimulatory molecules (ICAM-1,        B7-1 and B7-2) and are poor stimulators of unprimed T cells.        After contact with antigen, some Langerhans cells become        activated, exit the epidermis and migrate to T-cell-dependent        regions of regional lymph nodes where they local as mature        dendritic cells. In the course of exiting the epidermis and        migrating to lymph nodes, antigen-bearing epidermal Langerhans        cells (now the ‘messengers’) exhibit dramatic changes in        morphology, surface phenotype and function. In contrast to        epidermal Langerhans cells, lymphoid dendritic cells are        essentially non-phagocytic and process protein antigens        inefficiently, but express high levels of MHC class I and class        II antigens and various costimulatory molecules and are the most        potent stimulators of naive T cells that have been identified.”

We envision that the potent antigen presenting capability of theepidermal Langerhans cells can be exploited for transcutaneouslydelivered vaccines. A transcutaneous immune response using the skinimmune system would require delivery of vaccine antigen only toLangerhans cells in the stratum corneum (the outermost layer of the skinconsisting of cornified cells and lipids) via passive diffusion andsubsequent activation of the Langerhans cells to take up antigen,migrate to B-cell follicles and/or T-cell dependent regions, and presentthe antigen to B and/or T cells. If antigens other that bAREs (forexample diptheria toxoid) are to be phagocytosed by the Langerhanscells, then these antigens could also be taken to the lymph node forpresentation to T-cells and subsequently induce an immune responsespecific for that antigen (e.g., diptheria toxoid). Thus, a feature oftranscutaneous immunization is the activation of the Langerhans cell,presumably by bacterial ADP-ribosylating exotoxins, ADP-ribosylatingexotoxin binding subunits (e.g., cholera toxin B subunit), or otheradjuvants or Langerhans cell activating substance. Increasing the skinpopulation of Langerhans cells using strategies such as acetone swabbingcould then be expected to enhance the transcutaneous immune response.

The spectrum of more commonly known skin immune responses is representedby contact dermatitis and atopy. Contact dermatitis, a pathogenicmanifestation of LC activation, is directed by Langerhans cells whichphagocytose antigen, migrate to lymph nodes, present antigen, andsensitize T cells that migrate to the skin and cause the intensedestructive cellular response that occurs at affected skin sites (Dahl,1996; Leung, 1997). Atopic dermatitis may utilize the Langerhans cell ina similar fashion, but is identified with Th2 cells and is generallyassociated with high levels of IgE antibody (Dahl, 1996; Leung, 1997).

Transcutaneous immunization with cholera toxin and related bAREs on theother hand is a novel immune response with an absence of superficial andmicroscopic post-immunization skin findings (i.e., non-inflamed skin)shown by the absence of lymphocyte infiltration 24, 48 and 120 hoursafter immunization. This is strikingly shown by completion of a Phase Itrial in which humans were immunized with LT under a simple occlusivepatch. Potent anti-LT IgG and IgA antibodies were stimulated. Twovolunteers had biopsies performed at the site of immunization.Micro-scopic evaluation confirmed the clinical observation that noinflammation was seen. This suggests that Langerhans cells, which“comprise all of the accessory cell activity that is present inuninflammed epidermis, and in the current paradigm are essential for theinitiation and propagation of immune responses directed againstepicutaneously applied antigens” (Udey, 1997) may have been recruited.The uniqueness of the transcutaneous immune response here is alsoindicated by the both high levels of antigen-specific IgG antibody, andthe type of antibody produced (e.g., IgG1, IgG2a, IgG2b, IgG3 and IgA)and the absence of anti-CT IgE antibody. However, other immune cells maybe engaged and speculation on the mechanism should not limit theinvention.

Thus, we have found that bacterial-derived toxins applied to the surfaceof the skin can activate Langerhans cells and that TCI induces a potentimmune response manifested as high levels of antigen-specificcirculating IgG antibodies and would expect that penetration enhancementwould enhance the immune response. Transcutaneous adjuvant andpenetration enhancer may be used in transcutaneous immunization toenhance the IgG antibody or T-cell response to proteins not otherwiseimmunogenic by themselves when placed on the skin.

Transcutaneous targeting of Langerhans cells may also be used todeactivate their antigen presenting function, thereby preventingimmunization or sensitization. Techniques to mobilize Langerhans cellsor other skin immune cells yet negatively modulate them include, forexample, the use of anti-inflammatory steroidal or non-steroidal agents(NSAID), cyclophosphamide or other imnunosuppressants, interleukin-10,TGFβ monoclonal antibody to interleukin-1, ICE inhibitors or depletionvia superantigens such as through staphylococcal enterotoxin-A (SEA)induced epidermal Langerhans cell depletion.

Transcutaneous immunization may be induced via the ganglioside GM1binding activity of CT, LT or subunits such as CTB. Ganglioside GM1 is aubiquitous cell membrane glycolipid found in all mammalian cells. Whenthe pentameric CT B subunit binds to the cell surface a hydrophilic poreis formed which allows the A subunit to penetrate across the lipidbilayer.

We have shown that transcutaneous immunization by CT or CTB may requireganglioside GM1 binding activity. When mice are transcutaneouslyimmunized with CT, CTA and CTB, only CT and CTB resulted in an immuneresponse. CTA contains the ADP-ribosylating exotoxin activity but onlyCT and CTB containing the binding activity are able to induce an immuneresponse indicating that the B subunit was necessary and sufficient toimmunize through the skin. We conclude that the Langerhans cell or otherimmune cells may be activated by CTB binding to its cell surface, butmore activated by the concurrent presence of the A-subunit.

In addition to activation of the skin component in the immunizationprocess of the present invention, the antigen and/or adjuvant may beactivated to enhance immunization. When CT is secreted, cleavage occursat the trypsin recognition site and the toxin is activated. LT however,is secreted with its trypsin recognition site intact. When LT issecreted in the gastrointestinal tract and thereby exposed togastrointestinal agents such as trypsin, the proteolytically sensitiveresidues that join A1 and A2 subunits of LT are cleaved, allowing the A1subunit to ADP-ribosylate G proteins and therefore exert its toxiceffects. The lack of trypsin or related agents in the skin may preventtrypsin cleavage of the proteolytically sensitive residues that join A1and A2 subunits of LT, diminishing its adjuvant activity.

These two bacterial enterotoxins have many features in common. LT and CThave the same subunit number (A2:B5) and arrangement, and the samebiological mechanism of action. An amino acid sequence similarity of75-77% is found for both chains when comparing LT and CT, and the mostsignificant difference occurs in the respective A chains at positions192-195. At this site the cleavage of the A chain by trypsin occurs andthe site is situated between two cysteine residues that form theinternal disulfide bond of the A chain. See, for example, Mekalanos etal. (1979), Spangler (1992), and Sniderman (1995). We propose that thesestructural differences between the molecules are a significant influencenot only on their enterotoxic properties, but also on their ability tofunction as adjuvants.

Unlike CT produced by V. cholerae, LT is not fully biologically activewhen first isolated from the bacterial cell. Consistent with the A-Bmodel for bacterial toxins, LT requires trypsin proteolysis anddisulfide reduction to be fully active (Sniderman, 1995). In the absenceof proteolytic processing, the enzymatically active A1 moiety is unableto dissociate from the A2 component and cannot reach its targetsubstrate (adenylate cyclase) on the basolateral surface of theintestinal epithelial cell. This difference in activation of theisolated material results in differences in response thresholds for LTand CT in biologic systems. For instance, CT induces detectable netfluid secretion in the mouse intestine at a dose of 5 to 10 μg. LTinduces detectable net secretion in this assay at 50 to 100 μg. In therabbit ligated illeal loop, the difference is more dramatic and clearcut. Significantly however, when LT is exposed to proteolytic enzymeswith trypsin-like specificity, the molecule becomes indistinguishablefrom CT in any biologic assay system (Clements and Finkelstein, 1979;Dickenson and Clements, 1995).

According to Spangler (1992, citations omitted):

-   -   “Subunit A is synthesized as a single polypeptide in both V.        cholerae and E. coli. CTA is proteolytically “nicked” between        residues 192 and 195 during secretion from the vibrio by V.        cholerae hemagglutinin/protease, giving rise to two        polypeptides, A1 (Mr=28,826) and A2 (Mr=5,407), covalently        linked through a disulfide bridge between residues 187 and 199.        In contrast, LT remains in the E. coli periplasm and is not        nicked. Introduced into a genetically engineered strain of V.        cholerae, LT remained unnicked, although it was secreted in the        same manner as CT. Proteolytic processing is therefore not a        prerequisite for secretion. Purified LTh can, however, be nicked        in vitro, suggesting that the mutant vibrio used by Hirst et al.        contained insufficient soluble hemagglutinin to catalyze        nicking, rather than indicating an inability of LTA to be        nicked. CT, when introduced via an engineered plasmid into E        coli, remains unnicked and cell associated in E. coli.        Therefore, the defect in processing of CT and LT in E. coli is        related to the failure of E. coli to nick and secrete either        toxin. This defect may explain the reduced severity of E.        coli-induced enteric disease when compared with cholera. In both        CT and LT, the disulphide bond linking A1 to A2 remains        unreduced and the toxin is therefore essentially inactive, until        it enters a cell.    -   “Both the intact A subunit and the holotoxin are relatively        inactive ADP-ribosyltransferases compared with the A1        polypeptide. Catalytic activity requires the reduction of the        disulfide bond (A1:Cys-187-A2:Cys-199) linking A1 to A2. The        cleavage (nicking) between residues A1-Arg 192 and the start of        the A2 polypeptide at A2:Met-195 takes place during secretion of        CT from the vibrio. tryptic digestion serves the purpose in        vitro for LT. Reduction, which releases CTA1 from CTA2, may be        accomplished by a variety of agents, usually dithiothreitol or        2-mercaptoethanol in vitro or a thiol:protein oxireductase. The        endogenous reducing agent and mechanisms of reduction are not        known. An observed time lag of about 16 min between the apparent        binding of the toxin to the membrane receptor and the first        appearance of the modified substrate intracellularly may be        related to the time required for this step to occur following or        during insertion or translocation”.        LTh stands for LT holoezyme. Thus, if trypsin-treated LT were to        be used for transcutaneous immunization, we propose similar        mechanisms for disrupting disulphide bonds would occur. This may        be shown for trypsin activation of LT in which trypsin-activated        LT is similarly potent or of greater potentcy compared to CT and        much greater in potency to untreated LT in the mouse Y-1        bioassay (see Dickinson and Clements, 1995).

We propose to activate components of the formulation such as LT usingtrypsin or similar compounds prior to application the skin to enhancethe adjuvant activity and immunogenicity of LT. Activation of LT couldalso be expected to enhance the immune response to LT as an antigen. Theactivated adjuvant for transcutaneous immunization is preferably anADP-ribosylating exotoxin. Optionally, hydration or occlusive dressingsmay be used in the transcutaneous delivery system in addition to theactivation of the adjuvant.

In addition, LT has an unusual affinity for carbohydrate-containingmatrices. Specifically, LT binds to an array of biological moleculescontaining galactose, including glycoproteins and lipopolysaccharides.This lectin-like binding property of LT results in a broader receptordistribution on mammalian cells for LT than for CT, which binds only toGM1. The two molecules also have many immunologic differences, asdemonstrated by immunodiffusion studies, against LT-associated E. colidiarrhea in volunteers receiving B-subunit whole whole-cell choleravaccine. LT and CT induce different helper T-cell responses. When usedas a mucosal adjuvant, CT selectively induces in some cases Th2-typecells in Peyers patches and spleens as manifested by production ofinterleukins 4 and 5, but not interleukin 2 or gamma interferon; whileLT induces both Th1 and Th2 cells and predominantly antigen-specific IgAresponses. Taken together, these findings demonstrate that LT and CT areunique molecules, despite their apparent structural similarities. Suchdifferential behavior makes the ability to activate LT so that it haspotency similar to CT useful in manipulating the type of immune responseproduced to both the toxin itself and to antigens for which LT can beused as an adjuvant. It may also be possible that genetically alteredtoxoids such as mutants of the trypsin cleavage site may be active bytranscutaneous immunication. Such a mutant toxin may be useful as itavoids the risks associated with ingestion or inhaling native toxins.

In a similar manner, PT may be activated to enhance its adjuvant andantigen activities. The S1 subunit of the hexameric PT protein containsthe ADP-ribosyltransferase activity while the remaining subunitsconstitute the B domain. Similar to LT, PT has both trypsin cleavagesites and disulphide binding sites that play a role in association ofthe S1 subunit with the B oligomer. It is conceivable that activation bytrypsin cleavage, disruption of the disulphide bond or both may enhancethe adjuvant and antigen activities of PT in the context oftranscutaneous immunization. Activation may also take the form oftargeting, achieved by disruption of the hexamer into subunits. Forexample, the PT subunit S3 binds exclusively to the glycolipids ofmonocytes and could be used to target Langerhans cells in the skin.

Activation of the antigen or adjuvant could be extended to the conceptof transcutaneous immunization using DNA by production of a fusionprotein comprised of antigen and adjuvant domains. By this method aplasmid encoding an ADP-ribosylating exotoxin such as CT or LT andconstructed to express a separate antigen such as a malaria or HIVantigen simultaneously could be placed on the skin in a hydratingsolution or occlusive patch, and then taken up by Langerhans cells.Expression of the an ADP-ribosylating exotoxin component of the fusionsprotein such as CT or LT could activate the Langerhans cell, causing itto migrate and present antigen in the lymph node and thereby induce animmune response to the encoded antigen. Another embodiment could includethe conjugation of an adjuvant with a plasmid; an Fc portion of IgG to aplasmid to target APCs. A similar immunization could be achieved usingseparate plasmids for expressing an ADP-ribosylating exotoxin such as CTor LT and another for expressing the antigen such as a malaria or HIVantigen. It is conceivable that multiple genes on a single construct formultiple antigens could be used or multiple plasmids could be used tosimultaneously deliver antigens for multivalent immunization. Plasmidsencoding other molecules or compounds such as chemokines (e.g.,defensins 1 or 2, RANTES, MIP1-α, MIP-2, interleukin-8) or a cytokine(e.g., interleukin-1β, -2, -6, -10 or -12; γ-interferon; tumor necrosisfactor-α; or granulocyte-monocyte-colony stimulating factor) (reviewedin Nohria and Rubin, 1994), a heat shock protein or a derivative, aderivative of Leishmania major LeIF (Skeiky et al., 1995), cholera toxintoxin B, a lipopolysaccharide (LPS) derivative (e.g., lipid A ormonophosphoryl lipid A), or superantigen (Saloga et al., 1996), or otherADP-ribosylating exotoxins might be delivered with protein antigens.

Other means of activating the transcutaneous adjuvants may be effective,such as adding detergents and phospholipid to the formulation to enhanceCT activity by ADP-ribosylation factor (see, for example, Spangler,1992).

For immunization using adjuvant or antigen activation, modification ofthe adjuvant or antigen component of the formulation may reduce itseffectiveness in parenteral immunization without destroying the utilityof the formulation in transcutaneous immunization when the adjuvantand/or antigen is activated. Undesirable properties (e.g., toxicity,allergic reactivity, other side effects) of the adjuvant or antigen inthe formulation may be reduced by modification without destroying itseffectiveness in transcutaneous immunization. Activation of suchmodified adjuvant or antigen may involve, for example, removal of areversible chemical modification (e.g., proteolysis) or a coating whichreversibly isolates a component of the formulation from the immunesystem (i.e., an encapsulated formulation). Alternatively, the adjuvantand/or antigen comprising the formulation may be encapsulated in aparticle (e.g., microspheres, nanoparticles). Phagocytosis of a particlemay, by itself, enhance activation of an antigen presenting cell byupregulating expression of major histocompatibility antigens and/orcostimulatory molecules (e.g., MHC class II, B7-2).

Antigen

Antigen of the invention may be expressed by recombinant means,preferably as a fusion with an affinity or epitope tag (Summers andSmith, 1987; Goeddel, 1990; Ausubel et al., 1996); chemical synthesis ofan oligopeptide, either free or conjugated to carrier proteins, may beused to obtain antigen of the invention (Bodanszky, 1993; Wisdom, 1994).Oligopeptides are considered a type of polypeptide. Oligopeptide lengthsof 6 residues to 20 residues are preferred. Polypeptides may also besynthesized as branched structures such as those disclosed in U.S. Pat.Nos. 5,229,490 and 5,390,111. Antigenic polypeptides include, forexample, synthetic or recombinant B-cell and T-cell epitopes, universalT-cell epitopes, and mixed T-cell epitopes from one organism or diseaseand B-cell epitopes from another. Antigen obtained through recombinantmeans or peptide synthesis, as well as antigen of the invention obtainedfrom natural sources or extracts, may be purified by means of theantigen's physical and chemical characteristics, preferably byfractionation or chromatography (Janson and Ryden, 1989; Deutscher,1990; Scopes, 1993). A multivalent antigen formulation may be used toinduce an immune response to more than one antigen at the same time.Conjugates may be used to induce an immune response to multipleantigens, to boost the immune response, or both. Additionally, toxinsmay be boosted by the use of toxoids, or toxoids boosted by the use oftoxins. Transcutaneous immunization may be used to boost responsesinduced initially by other routes of immunization such as by oral, nasalor parenteral routes. Antigen includes, for example, toxins, toxoids,subunits thereof, or combinations thereof (e.g., cholera toxin, tetanustoxoid); additionally, toxins, toxoids, subunits thereof, orcombinations thereof may act as both antigen and adjuvant.

Antigen may be solubilized in a buffer. Suitable buffers include, butare not limited to, phosphate buffered saline Ca⁺⁺/Mg⁺⁺ free (PBS),normal saline (150 mM NaCl in water), and Tris buffer. Glycerol may be asuitable non-aqueous buffer for use in the present invention. Antigenmay also be in suspension. The detergent may be left in the immunizingsolution to enhance penetration.

Hydrophobic antigen can be solubilized in a detergent, for example apolypeptide containing a membrane-spanning domain. Furthermore, forformulations containing liposomes, an antigen in a detergent solution(e.g., a cell membrane extract) may be mixed with lipids, and liposomesthen may be formed by removal of the detergent by dilution, dialysis, orcolumn chromatography. See, Gregoriadis (1993). Certain antigens suchas, for example, those from a virus (e.g., hepatitis A) need not besoluble per se, but can be incorporated directly into a lipid membrane(e.g., a virosome as described by Morein and Simons, 1985), in asuspension of virion alone, or suspensions of microspheres nanoparticlesor heat-inactivated bacteria which may be taken up by and activateantigen presenting cells (e.g., opsonization).

Plotkin and Mortimer (1994) provide antigens which can be used tovaccinate animals or humans to induce an immune response specific forparticular pathogens, as well as methods of preparing antigen,determining a suitable dose of antigen, assaying for induction of animmune response, and treating infection by a pathogen (e.g., bacterium,virus, fungus, or parasite).

Bacteria include, for example: anthrax, campylobacter, cholera,clostridia, diphtheria, enterotoxigenic E. coli, giardia, gonococcus,Helicobacter pylori or urease produced by H. pylori (Lee and Chen,1994), Hemophilus influenza B, Hemophilus influenza non-typable,meningococcus, mycobacterium, pertussis, pneumococcus, salmonella,shigella, staphylococcus, Streptococcus B, tetanus, Vibrio cholerae,Borrelia burgdorfi and Yersinia; and products thereof.

Viruses include, for example: adenovirus, dengue serotypes 1 to 4(Delenda et al., 1994; Fonseca et al., 1994; Smucny et al., 1995), ebola(Jahrling et al., 1996), enterovirus, hanta virus, hepatitis serotypes Ato E (Blum, 1995; Katkov, 1996; Lieberman and Greenberg, 1996; Mast,1996; Shafara et al., 1995; Smedila et al., 1994; U.S. Pat. Nos.5,314,808 and 5,436,126), herpes simplex virus 1 or 2, humanimmunodeficiency virus (Deprez et al., 1996), human papilloma virus,influenza, measles, Norwalk, Japanese equine encephalitis, papillomavirus, parvovirus B19, polio, rabies, respiratory syncytial virus,rotavirus, rubella, rubeola, St. Louis encephalitis, vaccinia, vacciniaconstructs containing genes coding for other antigens such as malariaantigens, varicella, and yellow fever; and products thereof.

Parasites include, for example: Entamoeba histolytica (Zhang et al.,1995); Plasmodium (Bathurst et al., 1993; Chang et al., 1989, 1992,1994; Fries et al., 1992a, 1992b; Herrington et al., 1991; Khusmith etal., 1991; Malik et al., 1991; Migliorini et al., 1993; Pessi et al.,1991; Tam, 1988; Vreden et al., 1991; White et al., 1993; Wiesmueller etal., 1991), Leishmania (Frankenburg et al., 1996), and the Helminthes;and products thereof.

Other viruses which can be used in the present invention are disclosedin Gordon, 1997 and include, for example, Adenovirus (respiratorydisease), Coronavirus (respiratory and enteric disease), Cytomegalovirus(mononucleosis), Dengue virus (dengue fever, shock syndrome),Epstein-Barr virus (mononucleosis, Burkitt's lymphoma), Hepatitis A, Band C virus (liver disease), Herpes simplex virus type 1 (encephalitis,stomatitis), Herpes simplex virus type 2 (genital lesions), Humanherpesvirus-6 (unknown, possibly Kaposi's sarcoma), Humanimmunodeficiency virus types 1 and 2 (acquired immunodeficiencysyndrome-AIDS), Human T-cell lymphotropic virus type 1 (T-cellleukemia), Influenza A, B, and C (respiratory disease), Japaneseencephalitis virus (pneumonia, encephalopathy), Measles virus (subacutesclerosing panencephalitis), Mumps virus (meningitis, encephalitis),Papillomavirus (warts, cervical carcinoma), Parvovirus (respiratorydisease, anemia), Poliovirus (paralysis), Polyomavirus JC (multifocalleukoencephalopathy), Polyomavirus BK (hemorrhagic cystitis), Rabiesvirus (nerve dysfunction), Respiratory syncytial virus (respiratorydisease), Rhinovirus (common cold), Rotavirus (diarrhea), Rubella virus(fetal malformations), Vaccinia virus (generalized infection), Yellowfever virus (jaundice, renal and hepatic failure), Varicella zostervirus (chickenpox).

Other bacteria which can be used in the present invention are disclosedin Gordon, 1997 and include, for example, Bacillus anthracis (anthrax);Bordetella pertussis (whooping cough), Borrelia burgdorferi (lymedisease), Campylobacter jejuni (gastroenteritis), Chlamydia trachomatis(pelvic inflammatory disease, blindness), Clostridium botulinum(botulism), Corynebacterium dipththeriae (diphtheria), Escherichia coli(diarrhea, urinary tract infections), Haemophilus influenzae(pneumonia), Helicobacter pylori (gastritis, duodenal ulcer), Legionellapneumophila (Legionnaires' disease), Listeria monocytogenes (meningitis,sepsis), Mycobacterium leprae (leprosy), Mycobacterium tuberculosis(tuberculosis), Neisseria gonorrhoeae (gonorrhea), Neisseriameningitidis (sepsis, meningitis), Pseudomonas aeruginosa (nosocomialinfections), Pseudomonas aeruginosa (nosocomial infections), Rickettsia(Rocky Mountain spotted fever), Salmonella (typhoid fever,gastroenteritis), Shigella (dysentery), Staphylococcus aureus (impetigo,toxic shock syndrome), Streptococcus pneumoniae (pneumonia, otitismedia), Streptococcus pyogenes (Rheumatic fever, pharyngitis), Treponemapallidum (syphilis), Vibrio cholerae (cholera), Yersinia pestis (bubonicplague).

Other parasites which can be used in the present invention are disclosedin Gordon, 1997 and include, for example, African trypanosomes(trypanosomiasis), Entamoeba histolytica (amebic dysentery), Giardialamblia (diarrheal disease), Leishmania (lesions of the spleen, tropicalsores), Plasmodium (malaria), Microfilariae (filariasis), Schistosomes(schistosomiasis), Toxoplasma gondii (toxoplasmosis), Trichomonasvaginalis (vaginitis), Trypanosoma cruzi (Chagas disease).

Fungi which can be used in the present invention are disclosed inGordon, 1997 and include, for example, Candida albicans (mucosalinfections), Histoplasma (lung, lymph node infections), Pneumocystiscarinii (pneumonia in AIDS), Aspergillus fumigatis (aspergillosis).

Adjuvant

The formulation also contains an adjuvant, although a single moleculemay contain both adjuvant and antigen properties (e.g., cholera toxin)(Elson and Dertzbaugh, 1994). Adjuvants are substances that are used tospecifically or non-specifically potentiate an antigen-specific immuneresponse. Usually, the adjuvant and the formulation are mixed prior topresentation of the antigen but, alternatively, they may be separatelypresented within a short interval of time.

Adjuvants are usually products of bacteria, parasites or even viruses orbacteria, but may be derived from other natural or synthetic sources.Adjuvants include, for example, an oil emulsion (e.g., complete orincomplete Freund's adjuvant), a chemokine (e.g., defensins 1 or 2,RANTES, MIP1-α, MIP-2, interleukin-8) or a cytokine (e.g.,interleukin-1β, -2, -6, -10 or -12; γ-interferon; tumor necrosisfactor-α; or granulocyte-monocyte-colony stimulating factor) (reviewedin Nohria and Rubin, 1994), a muramyl dipeptide derivative (e.g.,murabutide, threonyl-MDP or muramyl tripeptide), a heat shock protein ora derivative, a derivative of Leishmania major LeIF (Skeiky et al.,1995), cholera toxin or cholera toxin B, bARES, a lipopolysaccharide(LPS) derivative (e.g., lipid A or monophosphoryl lipid A, syntheticlipid A analogues), or superantigen (Saloga et al., 1996) blockcopolymers or other polymers known in the art. Also, see Richards et al.(1995) for other adjuvants useful in immunization.

An adjuvant may be chosen to preferentially induce antibody or cellulareffectors, specific antibody isotypes (e.g., IgM, IgD, IgA1, IgA2,secretory IgA, IgE, IgG1, IgG2, IgG3, and/or IgG4), or specific T-cellsubsets (e.g., CTL, Th1, Th2 and/or T_(DTH)) (Munoz et al., 1990; Glennet al., 1995).

CpGs are among a class of structures which have patterns allowing theimmune system to recognize their pathogenic origins to stimulate theinnate immune response leading to adaptive immune responses (Medzhitovand Janeway, 1997). These structures are called pathogen-associatedmolecular patterns (PAMPs) and include lipopolysaccharides, teichoicacids, unmethylated CpG motifs, double stranded RNA and mannins, forexample.

PAMPs induce endogenous danger signals that can enhance the immuneresponse, act as costimulators of T-cell function and control theeffector function. The ability of PAMPs to induce these responses play arole in their potential as adjuvants and their targets are APCs such asmacrophages and dendritic cells. The antigen presenting cells of theskin could likewise be stimulated by PAMPs transmitted through the skin.For example, Langerhans cells, a type of dendritic cell, could beactivated by a PAMP in solution on the skin with a transcutaneouslypoorly immunogenic molecule and be induced to migrate and present thispoorly immunogenic molecule to T-cells in the lymph node, inducing anantibody response to the poorly immunogenic molecule. PAMPs could alsobe used in conjunction with other skin adjuvants such as cholera toixnto induce different costimulatory molecules and control differenteffector functions to guide the immune response, for example from a Th2to a Th1 response.

Cholera toxin is a bacterial exotoxin from the family ofADP-ribsoylating exotoxins (referred to as bAREs). Most bAREs areorganized as A:B dimer with a binding B subunit and an A subunitcontaining the ADP-ribosyltransferase. Such toxins include diphtheria,Pseudomonas exotoxin A, cholera toxin (CT), E. coli heat-labileenterotoxin (LT), pertussis toxin (PT), C. botulinum toxin C2, C.botulinum toxin C3, C. limosum exoenzyme, B. cereus exoenzyme,Pseudomonas exotoxin S, Staphylococcus aureus EDIN, and B. sphaericustoxin.

Cholera toxin is an example of a bARE that is organized with A and Bsubunits. The B subunit is the binding subunit and consists of aB-subunit pentamer which is non-covalently bound to the A subunit. TheB-subunit pentamer is arranged in a symmetrical doughnut-shapedstructure that binds to GM1-ganglioside on the target cell. The Asubunit serves to ADP ribosylate the alpha subunit of a subset of thehetero trimeric GTP proteins (G proteins) including the Gs protein whichresults in the elevated intracellular levels of cyclic AMP. Thisstimulates release of ions and fluid from intestinal cells in the caseof cholera.

Cholera toxin (CT) and its B subunit (CTB) have adjuvant properties whenused as either an intramuscular or oral immunogen (Elson and Dertzbaugh,1994; Trach et al., 1997). Heat-labile enterotoxin from E. coli (LT) is75-77% homologous at the amino acid level with CT and possesses similarbinding properties; it also appears to bind the GM1-ganglioside receptorin the gut and has similar ADP-ribosylating exotoxin activities. AnotherbARE, Pseudomonas exotoxin A (ETA), binds to the α2-macroglobulinreceptor-low density lipoprotein receptor-related protein (Kounnas etal., 1992). bAREs are reviewed by Krueger and Barbieri (1995). CT, CTB,LT, ETA and PT, despite having different cellular binding sites, arepotent adjuvants for transcutaneous immunization, inducing high levelsof IgG antibodies but not IgE antibodies. CTB without CT can also inducehigh levels of IgG antibodies. Thus, both bAREs and a derivative thereofcan effectively immunize when epicutaneouly applied to the skin in asimple solution.

All licensed vaccines require a level of antibody for approval—no otherimmune component such as T-cell proliferation is used. Protectionagainst the life-threatening infections diphtheria, pertussis, andtetanus (DPT) can be achieved by inducing high levels of circulatinganti-toxin antibodies. Pertussis may be an exception in that someinvestigators feel that antibodies directed to other portions of theinvading organism are necessary for protection, although this iscontroversial (see Schneerson et al., 1996) and most new generationacellular pertussis vaccines have PT as a component of the vaccine(Krueger and Barbieri, 1995). The pathologies in the diseases caused byDPT are directly related to the effects of their toxins and anti-toxinantibodies most certainly play a role in protection (Schneerson et al.,1996).

In general, toxins can be chemically inactivated to form toxoids whichare less toxic but remain immunogenic. We envision that the oneembodiment of transcutaneous immunization system will use toxin-basedimmunogens and adjuvants to achieve anti-toxin levels adequate forprotection against these diseases. The anti-toxin antibodies may beinduced through immunization with the toxins, or genetically-detoxifiedtoxoids themselves, or with toxoids and adjuvants such as CT.Genetically toxoided toxins which have altered ADP-ribosylating exotoxinactivity, or trypsin cleavage site mutations or other mutations areenvisioned to be especially useful as non-toxic activators of antigenpresenting cells used in transcutaneous immunization. Mutants based oninactivating the catalytic activity of the ADP-ribosyl transferase bygenetic deletion retain the binding capabilities, but lack the toxicity,of the natural toxins. This approach is described by Burnette et al.(1994), Rappuoli et al. (1995), and Rappuoli et al. (1996). Suchgenetically toxoided exotoxins could be useful for transcutaneousimmunization system in that they would not create a safety concern asthe toxoids would not be considered toxic. There are other geneticallyaltered toxins which have, for example, deletions of the trysin cleavagesite and use both non-toxic and immunogeneric on the skin. However,activation through a technique such as trypsin cleavage would beexpected to enhance the adjuvant qualities of LT through the skin whichis lacking inherent trypsin enzymes. Additionally, several techniquesexist to chemically toxoid toxins which can address the same problem(Schneerson et al., 1996). These techniques could be important forcertain applications, especially pediatric applications, in whichingested toxins (e.g., diphtheria toxin) might possibly create adversereactions.

Optionally, an activator of Langerhans cells may be used as an adjuvant.Examples of such activators include: an inducer of heat shock protein;contact sensitizer (e.g., trinitrochlorobenzene, dinitrofluorobenzene,nitrogen mustard, pentadecylcatechol); toxin (e.g., Shiga toxin, Staphenterotoxin B); lipopolysaccharide, lipid A, or derivatives thereof;bacterial DNA (Stacey et al., 1996); cytokine (e.g., tumor necrosisfactor-α, interleukin-1β, -10, -12); calcium ions in solution; calciumionophores, and chemokine (e.g., defensins 1 or 2, RANTES, MIP-1α,MIP-2, interleukin-8).

If an immunizing antigen has sufficient Langerhans cell activatingcapabilities then a separate adjuvant may not be required, as in thecase of CT which is both antigen and adjuvant. It is envisioned thatwhole cell preparations, live viruses, attenuated viruses, DNA plasmids,and bacterial DNA could be sufficient to immunize transcutaneously thenan adjuvant is present. It may be possible to use low concentrations ofcontact sensitizers or other activators of Langerhans cells to induce animmune response without inducing skin lesions.

Practical Aspects of Transcutaneous Immunization

Efficient immunization can be achieved with the present inventionbecause transcutaneous delivery of antigen may target the Langerhanscell. These cells are found in abundance in the skin and are efficientantigen presenting cells leading to T-cell memory and potent immuneresponses. Because of the presence of large numbers of Langerhans cellsin the skin, the efficiency of transcutaneous delivery may be related tothe surface area exposed to antigen and adjuvant. In fact, the reasonthat transcutaneous immunization is so efficient may be that it targetsa larger number of these efficient antigen presenting cells thanintramuscular immunization.

We envision the present invention will enhance access to immunization,while inducing a potent immune response. Because transcutaneousimmunization does not involve physical penetration of the skin and thecomplications and difficulties thereof, the requirements of trainedpersonnel, sterile technique, and sterile equipment are reduced.Furthermore, the barriers to immunization at multiple sites or tomultiple immunizations are diminished. Immunization by a singleapplication of the formulation is also envisioned, but boosting isgenerally needed. Needle free immunization is a priority for the WorldHealth Organization (WHO) because of the reuse of needles which causesneedle-borne disease.

Immunization may be achieved using epicutaneous application of a simplesolution of antigen and adjuvant impregnated in gauze under an occlusivepatch, or by using other patch technologies; creams, gels, immersion,ointments and sprays are other possible methods of application. Theimmunization could be given by untrained personnel, and is amenable toself-application. Large-scale field immunization could occur given theeasy accessibility to immunization. Additionally, a simple immunizationprocedure would improve access to immunization by pediatric patients andthe elderly, and populations in Third World countries.

For previous vaccines, their formulations were injected through the skinwith needles. Injection of vaccines using needles carries certaindrawbacks including the need for sterile needles and syringes, trainedmedical personnel to administer the vaccine, discomfort from theinjection, and potential complications brought about by puncturing theskin with the needle. Immunization through the skin without the use ofneedles (i.e., transcutaneous immunization) represents a major advancefor vaccine delivery by avoiding the aforementioned drawbacks.

Moreover, transcutaneous immunization may be superior to immunizationusing needles as more immune cells would be targeted by the use ofseveral locations targeting large surface areas of skin. Atherapeutically effective amount of antigen sufficient to induce animmune response may be delivered transcutaneously either at a singlecutaneous location, or over an area of intact skin covering multipledraining lymph node fields (e.g., cervical, axillary, inguinal,epitrochelear, popliteal, those of the abdomen and thorax). Suchlocations close to numerous different lymphatic nodes at locations allover the body will provide a more widespread stimulus to the immunesystem than when a small amount of antigen is injected at a singlelocation by intradermal subcutaneous or intramuscular injection.

Antigen passing through or into the skin may encounter antigenpresenting cells which process the antigen in a way that induces animmune response. Multiple immunization sites may recruit a greaternumber of antigen presenting cells and the larger population of antigenpresenting cells that were recruited would result in greater inductionof the immune response. It is conceivable that absorption through theskin may deliver antigen to phagocytic cells of the skin such as, forexample, dermal dendritic cells, macrophages, and other skin antigenpresenting cells; antigen may also be delivered to phagocytic cells ofthe liver, spleen, and bone marrow that are known to serve as theantigen presenting cells through the blood stream or lymphatic system.Langerhans cells, dendritic cells, and macrophages may be specificallytargeted using Fc receptor conjugated to or recombinantly produced as aprotein fusion with adjuvant; also, complement receptors (C3, C5) may beconjugated to or recombinantly produced as a protein fusion with proteinA or protein G to target surface immunoglobulin of B cells. The resultwould be targeted distribution of antigen to antigen presenting cells toa degree that is rarely, if ever achieved, by current immunizationpractices.

The transcutaneous immunization system may be applied directly to theskin and allowed to air dry; rubbed into the skin or scalp; held inplace with a dressing, patch, or absorbent material; immersion;otherwise held by a device such as a stocking, slipper, glove, or shirt;or sprayed onto the skin to maximize contact with the skin. Theformulation may be applied in an absorbant dressing or gauze. Theformulation may be covered with an occlusive dressing such as, forexample, AQUAPHOR (an emulsion of petrolatum, mineral oil, mineral wax,wool wax, panthenol, bisabol, and glycerin from Beiersdorf, Inc.),plastic film, COMFEEL (Coloplast) or vaseline; or a non-occlusivedressing such as, for example, DUODERM (3M) or OPSITE (Smith & Napheu).An occlusive dressing completely excludes the passage of water. Theformulation may be applied to single or multiple sites, to single ormultiple limbs, or to large surface areas of the skin by completeimmersion. The formulation may be applied directly to the skin.

Genetic immunization has been described in U.S. Pat. Nos. 5,589,466,5,593,972, and 5,703,055. The nucleic acid(s) contained in theformulation may encode the antigen, the adjuvant, or both. It wouldgenerally be expected that the immune response would be enhanced by thecoadministration of an adjuvant, for example, CT, LT or CpGs to thenucleic acid encoding for the antigen. The nucleic acid may or may notbe capable of replication; it may be non-integrating and non-infectious.For example, the nucleic acid may encode a fusion polypeptide comprisingantigen and a ubiquitin domain to direct the immune response to a classI restricted response. The nucleic acid may further comprise aregulatory region (e.g., promoter, enhancer, silencer, transcriptioninitiation and termination sites, RNA splice acceptor and donor sites,polyadenylation signal, internal ribosome binding site, translationinitiation and termination sites) operably linked to the sequenceencoding the antigen. The nucleic acid may be complexed with an agentthat promotes transfection such as cationic lipid, calcium phosphate,DEAE-dextran, polybrene-DMSO, or a combination thereof; also, immunecells can be targeted by conjugation of DNA to Fc receptor or proteinA/G, or encapsulating DNA in an agent linked to Fc receptor or proteinA/G. The nucleic acid may comprise regions derived from viral genomes.Such materials and techniques are described by Kriegler (1990) andMurray (1991).

An immune response may comprise humoral (i.e., antigen-specificantibody) and/or cellular (i.e., antigen-specific lymphocytes such as Bcells, CD4⁺ T cells, CD8⁺ T cells, CTL, Th1 cells, Th2 cells, and/orT_(DTH) cells) effector arms. Moreover, the immune response may compriseNK cells that mediate antibody-dependent cell-mediated cytotoxicity(ADCC).

The immune response induced by the formulation of the invention mayinclude the elicitation of antigen-specific antibodies and/or cytotoxiclymphocytes (CTL, reviewed in Alving and Wassef, 1994). Antibody can bedetected by immunoassay techniques, and the detection of variousisotypes (e.g., IgM, IgD, IgA1, IgA2, secretory IgA, IgE, IgG1, IgG2,IgG3, or IgG4) may be expected. An immune response can also be detectedby a neutralizing assay. Antibodies are protective proteins produced byB lymphocytes. They are highly specific, generally targeting one epitopeof an antigen. Often, antibodies play a role in protection againstdisease by specifically reacting with antigens derived from thepathogens causing the disease.

CTLs are particular protective immune cells produced to protect againstinfection by a pathogen. They are also highly specific. Immunization mayinduce CTLs specific for the antigen, such as a synthetic oligopeptidebased on a malaria protein, in association with self-majorhistocompatibility antigen. CTLs induced by immunization with thetranscutaneous delivery system may kill pathogen infected cells.Immunization may also produce a memory response as indicated by boostingresponses in antibodies and CTLs, lymphocyte proliferation by culture oflymphocytes stimulated with the antigen, and delayed typehypersensitivity responses to intradermal skin challenge of the antigenalone.

In a viral neutralization assay, serial dilutions of sera are added tohost cells which are then observed for infection after challenge withinfectious virus. Alternatively, serial dilutions of sera may beincubated with infectious titers of virus prior to innoculation of ananimal, and the innoculated animals are then observed for signs ofinfection.

The transcutaneous immunization system of the invention may be evaluatedusing challenge models in either animals or humans, which evaluate theability of immunization with the antigen to protect the subject fromdisease. Such protection would demonstrate an antigen-specific immuneresponse. In lieu of challenge, for example achieving anti-diphtheriaantibody titers of 5 IU/ml or greater is generally assumed to indicateoptimum protection and serves as a surrogate marker for protection(Plotkin and Mortimer, 1994).

Vaccination has also been used as a treatment for cancer and autoimmunedisease. For example, vaccination with a tumor antigen (e.g., prostatespecific antigen) may induce an immune response in the form ofantibodies, CTLs and lymphocyte proliferation which allows the body isimmune system to recognize and kill tumor cells. Tumor antigens usefulfor vaccination have been described for melanoma (U.S. Pat. Nos.5,102,663, 5,141,742, and 5,262,177), prostate carcinoma (U.S. Pat. No.5,538,866), and lymphoma (U.S. Pat. Nos. 4,816,249, 5,068,177, and5,227,159). Vaccination with T-cell receptor oligopeptide may induce animmune response that halts progression of autoimmune disease (U.S. Pat.Nos. 5,612,035 and 5,614,192; Antel et al., 1996; Vandenbark et al.,1996). U.S. Pat. No. 5,552,300 also describes antigens suitable fortreating autoimmune disease.

The following is meant to be illustrative of the present invention;however, the practice of the invention is not limited or restricted inany way by the examples.

EXAMPLES

Immunization procedure. Twenty four hours prior to immunization, theback of the mouse is shaved from the distal aspect of the scapula to 0.5cm above the base of the tail. In the case of C57BL/6 mice, the animalsare lightly anesthetized (40 mg/kg ketamine:4 mg/kg xylazine mixture insaline) prior to shaving. On the day of immunization the animals areimmunized with 0.04 ml of an anesthesia mixture (2.3 mL sterile saline(Sigma): 5 mL ketamine (100 mg/mL, Parke-Davis): 0.5 mL xylazine (100mg/mL, Phoenix Pharmaceuticals)) which delivers a final dose ofapproximately 110 mg/kg ketamine and 11 mg/kg xylazine. For proceduresrequiring alcohol swabbing, the back is wiped 10× (5× sweeping up theback towards the head, flip over alcohol pad and sweep back 5× more)using an isopropyl pad. The alcohol is allowed to evaporate for 5minutes. Hydration of the back is accomplished by gently rubbing theback with a sterile water-saturated gauze pad so as to form a pool ofwater on the back. After a 5 minute hydration period, the back isblotted dry with a dry gauze pad. Next, antigen—generally ≦100 μg ofantigen and adjuvant in 100 μl final volume, is applied to the backusing a pipette and tip and left on the skin for 60 to 120 minutes.After the defined immunization period has been reached, any excesssolution in the immunized area is blotted with cotton gauze. The animalsare then rinsed animals under a slow steady stream of lukewarm tap waterfor 10 seconds to remove any excess antigen, blotted dry and the rinsingprocedure repeated. The cages are then placed onto the heating padsuntil they are fully recovered from the anesthesia.Measurement of Human anti-LT Antibody Titers. Anti-LT IgG titers weredetermined as previously described (Svennerholm A-M., Holmgren, J.,Black, R., Levine, M. & Merson, M. Serologic differentation betweenantitoxin responses to infection with Vibrio cholerae andenterotoxin-producing Escherichia coli. J. Infect. Dis. 147, 541-522(1983). 96 well (Type-Russell) plates were coated overnight withmonosialoganglioside-G_(Ml) (Sigma, St. Louis, Mo.) of LT (Sigma),blocked with 5% dry milk in PBS-0.05% Tween. Responses were detectedusing goat anti-human IgG(γ)-HRP (Kirkegaard and Perry, Gaithersburg,Md., and 2,2′-azino-di[3-ethylbenzthiazoline sulfonate (Kirkegaard andPerry) as substrate and plates were read at 405 nm. Results are reportedin ELISA units (EU) which are defined as the inverse dilution of samplewhich yields an OD of 1.0. Anti-LT IgA was determined in the same manneras anti-LT IgG except that goat anti-human IgA(α)-HRP (Kirkegaard andPerry) was used as secondary antibody and ODs were plotted against astandard IgA curve yielding results expressed in ng/ml. The standard IgAcurve and total serum IgA were determined by using unlabeled goatanti-human IgA (Kirkegaard and Perry) followed by blocking as above andthen application of serial dilutions of IgA standard

Example 1

Swabbing the skin with a treated or untreated swab is thought tophysically and chemically remove a small portion of the stratum corneumand thus enhance skin penetration. Swabs can be made of materials suchas, for example, cotton, nylon, rayon and polyethylene. Alcohol swabbingis thought to remove a small portion of the stratum corneum and actsboth as a physical means and chemical means of penetration enhancement.In the example above, the enhancement of the immune response totranscutaneous immunization can be seen with this penetrationenhancement method. BALB/c mice 6 to 8 weeks of age were anesthetizedand shaved as described in the “immunization procedure”. Twenty fourhours later, the backs of the animals were either wiped with a gauze padsaturated in water “water” or wiped for approximately 10 seconds with analcohol prep pad containing 70% isopropyl alcohol “isopropanol”. Thealcohol was allowed to evaporate for approximately 5 minutes. The excesswater was removed from the backs of the “water” group by blotting. Allanimals were then treated with 20 μg of CT (100 μl of a 0.2 mg/mlsolution). Removal of excess antigen was conducted as described in the“immunization procedure.”

The anti-CT antibody titers were determined using ELISA as describedabove for “ELISA IgG (H+L)” 3 weeks after a single immunization. Theresults are shown in Table 1. While CT was clearly immunogenic in bothgroups, the group treated with the alcohol prep pads exhibited ageometric mean titer that was 6 fold higher and the individual titerswere more consistent than the “water” animals. Thus it appears thatchemical and physical disruption of the skin surface with alcohol swabsenhances delivery of antigen by the transcutaneous route.

TABLE 1 Enhancement of transcutaneous immunization by chemicalpenetration enhancement: Anti-CT titers in mice that had the skintreated with an alcohol prep pad before application of the antigen.anti-CT IgG (H + L) ELISA units Animal # treatment prebleed week 3 7146water 1275 7147 water 69 7148 water 7420 7149 water 6025 7150 water 388geometric mean 1088 pooled prebleed 7 7161 isopropanol 3100 7162isopropanol 14797 7163 isopropanol 6670 7164 isopropanol 7426 7165isopropanol 7024 geometric mean 6928 pooled prebleed 7

Example 2

To assess whether chemical penetration enhancement alone might augmenttranscutaneous immunization a detergent was used on the skin. BALB/cmice 6 to 8 weeks of age were anesthetized and shaved as described inthe “immunization procedure.” Twenty-four hours later, the backs of the“water” group were wiped with a gauze pad saturated in water and a poolof water was placed on the back. Approximately 5 minutes later, anyexcess water was removed and 25 μg of CT (50 μl of a 0.5 mg/ml solution)was applied to the back. Alternatively, 24 hours after shaving, thebacks of the “5% SDS” group were treated by dripping 300 μl of 5% SDS(Sodium Dodecyl Sulfate—a 1 to 1 mixture of deionized water andcommercial stock of 10% SDS), a detergent, for approximately 12 minutesfollowed by blotting off any excess SDS with a dry gauze pad. SDS can beapplied to the skin in a carrier such as, for example, a pad and thenany excess SDS can be removed with a dry gauze pad. Thereafter theanimals were hydrated and immunized as per the “water” group. Removal ofexcess antigen was conducted as described in the “immunizationprocedure.”

The anti-CT antibody titers were determined using ELISA as describedabove for “ELISA IgG (H+L)” 2 weeks after a single immunization. Theresults are shown in tables 2a and 2b. While CT was clearly immunogenicin both groups, the geometric mean titer in the 5% SDS treated groupapproximately 2 fold higher and the titers were more consistent amongthe latter animals as compared with the “water” animals. Thus it appearsthat chemical disruption of the skin surface with detergent (5% SDS)enhances delivery of antigen by the transcutaneous route.

TABLE 2a Enhancement of transcutaneous immunization by chemicalpenetration enhancement: Anti-CT titers in mice that had the skintreated with detergent (5% SDS) before application of the antigen.anti-CT IgG (H + L) ELISA units Animal # treatment prebleed week 2 546water 4629 547 water 3154 548 water 7288 549 water 1719 550 water 11779geometric mean 3678 pooled prebleed 5 596 5% SDS 6945 597 5% SDS 2244598 5% SDS 8604 599 5% SDS 7093 600 5% SDS 12583 geometric mean 5553pooled prebleed 1

TABLE 2b Enhancement of transcutaneous immunization by chemicalpenetration enhancement: Anti-CT titers in mice that had the skintreated with detergent (5% SDS) before application of the antigen.anti-CT IgG (H + L) ELISA units Animal # treatment prebleed week 3 546water 22525 547 water 8939 548 water 11885 549 water 5121 550 water37770 geometric mean 10521 pooled prebleed 11 596 5% SDS 102387 597 5%SDS 6597 598 5% SDS 47245 599 5% SDS 45565 600 5% SDS 38413 geometricmean 34725 pooled prebleed 6

Example 3

Another form of chemical penetration enhancement, a depilatory (such as,for example, calcium hydroxide or the like) is widely used indermatologic experiments and was shown to enhance transcutaneousimmunization. BALB/c mice 6 to 8 weeks of age were anesthetized andshaved as described in the “immunization procedure.” Twenty-four hourslater, the backs of the “water” group were wiped with a gauze padsaturated in water and a pool of water was placed on the back.Approximately 5 minutes later, any excess water was removed and 25 μg ofCT (50 μl of a 0.5 mg/ml solution) was applied to the back.Alternatively, twenty-four hours after shaving, the backs of the “nair©”group were treated with 100 μl of nair© cream for approximately 12minutes followed by wiping off of the formulation with a gauze padsaturated in water. Such treatment can continue for from about 0.1 to 30minutes preferably about 20 minutes and more preferably about 12minutes. Thereafter the animals were hydrated and immunized as per the“water” group. Removal of excess antigen was conducted as described inthe “immunization procedure.”

The anti-CT antibody titers were determined using ELISA as describedabove for “ELISA IgG (H+L)” 2 weeks after a single immunization. Theresults are shown in tables 3a and 3b. While CT was clearly immunogenicin both groups, the geometric mean titer in the nair treated group was 3fold higher and the titers were more consistent among the latter animalsas compared with the “water” animals. Thus it appears that chemicaldisruption of the skin surface with calcium hydroxide, the activeingredient in nair© cream, enhances delivery of antigen by thetranscutaneous route.

TABLE 3a Enhancement of transcutaneous immunization by chemicalpenetration enhancement: Anti-CT titers in mice that had the skintreated with calcium hydroxide (nair ©) before application of theantigen. anti-CT IgG (H + L) ELISA units Animal # treatment prebleedweek 2 546 water 4629 547 water 3154 548 water 7288 549 water 1719 550water 11779 geometric mean 3678 pooled prebleed 5 581 nair © 17621 582nair © 12261 583 nair © 7235 584 nair © 7545 585 nair © 5997 geometricmean 10421 pooled prebleed 4

TABLE 3b Enhancement of transcutaneous immunization by chemicalpenetration enhancement: Anti-CT titers in mice that had the skintreated with calcium hydroxide (nair ©) before application of theantigen. anti-CT IgG (H + L) ELISA units Animal # treatment prebleedWeek 3 546 water 22525 547 water 8939 548 water 11885 549 water 5121 550water 37770 geometric mean 10521 pooled prebleed 11 581 nair © 34222 582nair © 45674 583 nair © 50224 584 nair © 27270 585 nair © 21832geometric mean 38251 pooled prebleed 15

Example 4

Further studies were conducted to evaluate the effect of chemicalpenetration enhancement using a keratinolytic formulation (such as asalicylate). BALB/c mice 6 to 8 weeks of age were anesthetized andshaved as described in the “immunization procedure.” Twenty-four hourslater, the backs of the “water” group were wiped with a gauze padsaturated in water and a pool of water was placed on the back.Approximately 5 minutes later, any excess water was removed and 25 μg ofCT (50 μl of a 0.5 mg/ml solution) was applied to the back.Alternatively, twenty-four hours after shaving, the backs of the“salicylate/water” group were treated with a gauze pad saturated with a10% salicylate suspension (1 tablet (325 mg) Certified brand aspirindissolved in 3.25 ml deionized water). Such treatment can continue forfrom about 0.1 to 30 minutes preferably about 20 minutes and morepreferably about 10 minutes. Approximately 10 minutes later anyremaining solution was blotted off, the backs of the animals werehydrated with water for 5 minutes, followed by removal of excess water,and then topical application of 25 μg of CT. Removal of excess antigenwas conducted as described in the “immunization procedure.”

The anti-CT antibody titers were determined using ELISA as describedabove for “ELISA IgG (H+L)” 2 weeks after a single immunization. Theresults are shown in Table 4. While CT was clearly immunogenic in bothgroups, the geometric mean titer in the salicylate treated group was 4fold higher and the titers were more consistent among the latter animalsas compared with the “water” animals. Thus it appears that chemicaldisruption of the skin surface with salicylate enhances delivery ofantigen by the transcutaneous route.

TABLE 4 Enhancement of transcutaneous immunization by chemicalpenetration enhancement: Anti-CT titers in mice that had the skintreated with salicylate (aspirin) before application of the antigen.anti-CT IgG (H + L) ELISA units Animal # treatment prebleed week 2 741water 272 742 water not available 743 water 456 744 water 443 745 water1395 geometric mean 526 pooled prebleed 7 756 salicylate/water 2279 757salicylate/water 4581 758 salicylate/water 4658 759 Salicylate/water2771 760 Salicylate/water 593 geometric mean 2402 pooled prebleed 36

Example 5

To assess the role of physical/mechanical penetration enhancement, anabrasive, in the form of a common emory board, was used to remove aportion of the stratum corneum. BALB/c mice 6 to 8 weeks of age wereanesthetized and shaved as described in the “immunization procedure.”Twenty four hours later, the backs of the animals were either wiped witha gauze pad saturated in water “water” or brushed 10 times with a mediumgrain emory board “emory board.” and then wiped with a gauze padsaturated in water Approximately five minutes after the water treatment,any excess water was removed and 20 μg of CT (100 μl of a 0.2 mg/ml)solution applied to the back. Removal of excess antigen was conducted asdescribed in the “immunization procedure.”

The anti-CT antibody titers were determined using ELISA as describedabove for “ELISA IgG (H+L)” 3 weeks after a single immunization. Theresults are shown in Table 5. While CT was clearly immunogenic in bothgroups, the geometric mean titer in the emory board treated group was 10fold higher and the titers were more consistent among the latter animalsas compared with the “water” animals. Thus it appears that physicaldisruption of the outer surface of the skin with an emory board enhancesdelivery of antigen by the transcutaneous route. This can bedifferentiated from techniques that seek to pierce the skin and deliverantigen through the skin, such as in subcutaneous, intradermal orintramuscular injection.

This simple device could be replaced by other physical disruptingdevices to deliver antigens and adjuvants into the epidermis such asmicroneedles that are of length to disrupt only the stratum corneum orsuperficial epidermis, devices used for TB tine testing, gas poweredguns which do not penetrate the dermis, adhesive tape for tapestripping, or other barrier disruption devices known to disrupt only thestratum corneum or superficial epidermis.

TABLE 5 Enhancement of transcutaneous immunization by physicalpenetration enhancement: Anti-CT titers in mice that had the skintreated with an emory board before application of the antigen. anti-CTIgG (H + L) ELISA units Animal # treatment prebleed week 3 7146 water1275 7147 water 69 7148 water 7420 7149 water 6025 7150 water 388geometric mean 1088 pooled prebleed 7 7151 emory board 6632 7152 emoryboard 9380 7153 emory board 31482 7154 emory board 11142 7155 emoryboard 11761 geometric mean 12074 pooled prebleed 9

Example 6

Another means of physical/mechanical penetration enhancement wasemployed using an abrasive pad to remove a portion of the stratumcorneum and allow access to the underlying epidermis. BALB/c mice 6 to 8weeks of age were anesthetized and shaved as described in the“immunization procedure”. Twenty four hours later, the backs of theanimals were either wiped with a gauze pad saturated in water, “water”,or wiped with a gauze pad saturated in water followed by rubbing for 10seconds with a nylon sponge (buf puf ©) to remove the outermost layersof the stratum corneum, “buf-puf ©”. Excess water was removed from thebacks of the “water” group and then 20 μg of CT (100 μl of a 0.2 mg/mlsolution) was applied to the backs of all animals. Removal of excessantigen was conducted as described in the “immunization procedure.”

The anti-CT antibody titers were determined using ELISA as describedabove for “ELISA IgG (H+L)” 3 weeks after a single immunization. Theresults are shown in Table 6. While CT was clearly immunogenic in bothgroups, the geometric mean titer in the buff puff treated group was 2fold higher and the titers among individual animals were more consistentamong the latter animals compared with the “water” animals. Thus itappears that physical disruption of the skin surface with a buff-puf ©enhances delivery of antigen by the transcutaneous route.

This simple device could be replaced by other physical penetrationdevices to deliver antigens and adjuvants into the epidermis such as aneedle and tuberculin syringe used for intradermal injection,microneedles that are of length to penetrate only the stratum corneum orsuperficial dermis, devices used for TB tine testing, abrading patcheswhich have dissolvable crystals such as sucrose or sodium chloride orbiodegradable polymers that are impregnated into the patch and rubbed onthe skin before securing the patch with antigen either contained in thecrystal or in the matrix, gas powered guns, adhesive tape for tapestripping, or other devices known to penetrate only into the epidermisor superficial dermis.

TABLE 6 Enhancement of transcutaneous immunization by physicalpenetration enhancement: Anti-CT titers in mice that had the skintreated with an abrasive pad (such as, for example, buf-puf ©) beforeapplication of the antigen. anti-CT IgG (H + L) ELISA units Animal #treatment prebleed week 3 7146 water 1275 7147 water 69 7148 water 74207149 water 6025 7150 water 388 geometric mean 1088 pooled prebleed 77166 buf puf © 5376 7167 buf puf © 2319 7168 buf puf © 1209 7169 bufpuf © 2871 7170 buf puf © 2785 geometric mean 2607 pooled prebleed 8

Example 7

Transcutaneous immunization with bacterial ADP-ribosylating exotoxinssuch as CT and LT appear to provide significant ‘danger’ signals to theimmune system stimulating a potent immune response. Such compounds actas adjuvants. It was a surprise to find that simple mixtures of suchadjuvants placed on the skin in a manner that hydrates the skin,resulting in potent immune responses. This was described in earlierpatents (PCT/US97/21324). However, given that an adjuvant such as CT (86KD) can act as an adjuvant on the skin, it would be expected that otheradjuvants, particularly those based on bacterial products or motifs,would be stimulatory when placed on the skin in a manner that hydratesthe skin and/or with the use of penetration enhancers.

We used bacterial DNA to confirm that this expectation is correct.BALB/c mice 6 to 8 weeks of age were shaved and anesthetized asdescribed above for the “immunization procedure”. On the day ofimmunization the backs of the mice were wiped with isopropanol toenhance penetration. After the alcohol had evaporated (approximately 5minutes), 100 μl of phosphate buffered saline (PBS) containing 100 μg ofDNA (CpG1 or CpG2) and 100 μg of diphtheria toxoid (DT) was applied tothe back for 90 to 120 minutes. Oligonucleotides were synthesized byOligos Etc with a phosphorothioate backbone to improve stability.Removal of excess antigen was conducted as described in the“immunization procedure.” The immunization was repeated 4 and 8 weekslater. Ten weeks after the primary immunization the animals were bledand the anti-DT titers determined using an ELISA as described above for“ELISA IgG (H+L)”. The results are shown in Table 7A.

Co-administration of DT and a control DNA sequence (SEQ ID NO:1; CpG2:TCCAATGAGCTTCCTGAGTCT) failed to induce a detectable rise in the anti-DTtiters. In contrast, addition of a DNA sequence containing anunmethylated CpG dinucleotide flanked by two 5′ purines and two 3′pyrimidines (SEQ ID NO:2; CpG1 (immunostimulatory DNA):TCCATGACGTTCCTGACGTT) resulted in a detectable increase in the serumanti-DT IgG titer in 5 of 5 animals. Thus it appears that bacterial DNAcontaining appropriate motifs such as CPGs (6 KD) can be used asadjuvant to enhance delivery of antigen through the skin for inductionof antigen specific antibody responses.

TABLE 7A Adjuvant activity of bacterial DNA applied to the skin usingpenetration enhancement: humoral immune response. Anti-DT IgG (H + L)ELISA units Animal # adjuvant/antigen prebleed week 10 7261 CpG1/DT 11717262 CpG1/DT 22750 7263 CpG1/DT 4124 7264 CpG1/DT 126 7265 CpG1/DT 115Geometric mean 1096 pooled prebleed 6 7266 CpG2/DT 19 7267 CpG2/DT 127268 CpG2/DT 5 7269 CpG2/DT 5 7270 CpG2/DT 11 geometric mean 9 pooledprebleed 5

The transcutaneous effect of transcutaneous immunization can also bedetected by T-cell proliferation. BALB/c mice 6 to 8 weeks of age wereshaved and anesthetized as described above for the “immunizationprocedure”. On the day of immunization the backs of the mice were wipedwith isopropanol. After the alcohol had evaporated (approximately 5minutes), 100 μl of phosphate buffered saline (PBS) containing 100 μg ofDNA (CpG1 or CpG2) and 100 μg of diphtheria toxoid (DT) was applied tothe back for 90 to 120 minutes. Oligonucleotides were synthesized byOligos Etc with a phosphorothioate backbone to improve stability.Removal of excess antigen was conducted as described in the“immunization procedure.” The immunization was repeated 4 and 8 weekslater. Twelve weeks after the primary immunization draining (inguinal)LNs were removed and pooled from five immunized animals. The capacity toproliferate in response to media or antigen (DT) was assessed in astandard 4 day proliferation assay using 3-H incorporation as a readout.The results are shown in Table 7B. Co-administration of DT and a DNAsequence containing an unmethylated CpG dinucleotide flanked by two 5′purines and two 3′ pyrimidines (SEQ ID NO:2) resulted in a detectableincrease in the antigen specific proliferative response. Thus it appearsthat bacterial DNA containing appropriate motifs can be used as adjuvantto enhance delivery of antigen through the skin for induction ofproliferative responses.

TABLE 7B Adjuvant effect of bacterial DNA applied to the skin: LN cellproliferation proliferation (cpm) 3-H incorporation in vitro to antigensantigens applied in vivo media DT normal LN 339 544 CpG1/DT 1865 5741

Example 8

Transcutaneous immunization with bacterial ADP-ribosylating exotoxinssuch as CT and LT appear to provide significant ‘danger’ signals to theimmune system stimulating a potent immune response. Such compounds actas adjuvants. It was a surprise to find that simple mixtures of suchadjuvants placed on the skin in a manner that hydrates the skin,resulting in potent immune responses. This was described in earlierpatents (PCT/US97/21324). However, given that an adjuvant such as CT canact as an adjuvant on the skin, it would be expected that otheradjuvants, would be stimulatory when placed on the skin in a manner thathydrates the skin. Genetically altered toxins were used to confirm thisexpectation. BALB/c mice 6 to 8 weeks of age were anesthetized, shaved,and immunized as described in the “immunization procedure”. The animalswere boosted 3 and 5 weeks after the primary immunization and serumcollected 2 weeks after the final immunization. The adjuvants used werethe genetically altered toxins; LTK63, an enzymatically inactive LTderivative, and LTR72, an LT derivative which retains 0.6% of theenzymatic activity. Diphtheria toxoid (DT) 100 μg was used as antigen.

Anti-DT antibody titers were determined using ELISA as described abovefor “ELISA IgG (H+L)”. The results are shown in Table 8. Anti-DT titerswere clearly elevated in serum from animals immunized with either LTR63or LTR72 and DT when compared with titers in serum collected prior toimmunization (prebleed). Thus it appears that genetically detoxifiedmutants of heat labile enterotoxin (LT) can be used as adjuvants on theskin.

TABLE 8 Use of genetically altered toxins, LTK63 and LTR72, as adjuvantson the skin. anti-DT IgG (H + L) ELISA units Animal # adjuvant/antigenprebleed week 7 653 LTK63/DT 20228 654 LTK63/DT not available 655LTK63/DT 342 656 LTK63/DT 2445 657 LTK63/DT <100 geometric mean 1140pooled prebleed <100 663 LTR72/DT 12185 664 LTR72/DT 10917 665 LTR72/DT151 666 LTR72/DT 2057 667 LTR72/DT 50923 geometric mean 4620 pooledprebleed <100

Example 9

Another class of compounds, cytobines which are known to act asadjuvants illustrate the principle that adjuvants in general could beexpected to act in a fashion similar to cholera toxin. TNF∝ is alsoknown to be a Langerhan cell activating compound.

BALB/c mice 6 to 8 weeks of age were shaved and anesthetized asdescribed above for the “immunization procedure”. On the day ofimmunization the backs of the mice were wiped with isopropanol. Afterthe alcohol had evaporated (approximately 5 minutes), 100 μl ofphosphate buffered saline (PBS) containing 0.83 μg TNF-alpha(recombinant mouse TNF-alpha, Endogen), IL-2 (1 μg recombinant mouseIL-2 (Sigma)) or mock adjuvant (CpG2) was applied to the skin on theback with 100 μg of diphtheria toxoid (DT) for 90 to 120 minutes.Oligonucleotides were synthesized by Oligos Etc with a phosphorothioatebackbone to improve stability Removal of excess antigen was conducted asdescribed in the “immunization procedure.” The immunization was repeated4 and 8 weeks later. Ten weeks after the primary immunization theanimals were bled and the anti-DT titers determined using an ELISA asdescribed above for “ELISA IgG (H+L)”. The results are shown in Table 9.

Co-administration of DT and a mock adjuvant (CpG2) failed to induce adetectable rise in the anti-DT titers. In contrast, topical applicationof TNF-alpha (0.8 μg) resulted in a detectable increase in the serumanti-DT IgG titer in 3 of 5 animals when compared with either anti-DTtiters in the mock adjuvant treated mice or sera collected prior toimmunization (prebleed). Similarly, topical application of IL-2 (1 μg)resulted in a detectable increase in the serum anti-DT IgG titer in 4 of5 animals when compared with either anti-DT titers in the mock adjuvanttreated mice or sera collected prior to immunization (prebleed). Thus itappears that the cytokines such as IL-2 and TNF-alpha can be used as anadjuvant on the skin and that langerhans cell activating compounds canbe used for transcutaneous immunization.

TABLE 9 Adjuvant activity of the cytokine TNF-alpha applied to the skin.Anti-DT IgG (H + L) ELISA units Animal # adjuvant/antigen prebleed week10 7326 TNF-alpha/DT 1808 7327 TNF-alpha/DT 830 7328 TNF-alpha/DT 7 7329TNF-alpha/DT 1477 7330 TNF-alpha/DT 7 geometric mean 159 pooled prebleed1 7331 IL-2/DT 13 7332 IL-2/DT 111 7333 IL-2/DT 345 7334 IL-2/DT 49 7335IL-2/DT 35 geometric mean 61 pooled prebleed 2 7266 CpG2/DT 19 7267CpG2/DT 12 7268 CpG2/DT 5 7269 CpG2/DT 5 7270 CpG2/DT 11 geometric mean9 pooled prebleed 5

Example 10

The B-subunit of cholera toxin is another class of adjuvants lack theA-subunit and therefore ADP-ribsyltransferase activity of CT. As suchCTB represents an adjuvant that is unique and may be useful as it is nottoxic when ingested.

C57BL/6 mice 6 to 8 weeks of age were anesthetized and shaved asdescribed in the “immunization procedure”. On the day of immunizationthe backs of the mice were wiped with isopropanol. After the alcohol hadevaporated (approximately 5 minutes), 100 μl of phosphate bufferedsaline (PBS) containing 100 μg of purified cholera toxin B subunit (CTB)and/or 100 μg of diphtheria toxoid (DT) was applied to the back for 90to 120 minutes. Removal of excess antigen was conducted as described inthe “immunization procedure.” The immunization was repeated 4 and 8weeks later. Ten weeks after the primary immunization the animals werebled and the anti-DT titers determined using an ELISA as described abovefor “ELISA IgG (H+L)”. The results are shown in Table 10.

Anti-DT titers were clearly elevated in serum from animals immunizedwith CTB and DT when compared with titers in serum from animals treatedwith DT alone or those in prebleed serum samples as shown in Table 10.Thus it appears that purified CTB can be used as an adjuvant on theskin.

TABLE 10 Use of purified cholera toxin B subunit from V. cholerae as anadjuvant on the skin. Anti-DT IgG (H + L) ELISA units Animal #adjuvant/antigen prebleed week 10 51 DT 11 52 DT 7 53 DT 4 54 DT 8 55 DT7 geometric mean 7 pooled prebleed 4 81 CTB/DT 14880 82 CTB/DT 371 83CTB/DT 14810 84 CTB/DT 108 85 CTB/DT 27 geometric mean 751 pooledprebleed 5

Example 11

Adjuvants that are structurally different are likely to exert theirenhancement by different effects. Adjuvants that induce their effects bydifferent mechanisms may have either additive or synergistic effects onenhancing the immune response. We found that the use of two adjuvantssimultaneously augmented the response to transcutaneous immunizationcompared to the individual adjuvants alone.

BALB/c mice 6 to 8 weeks of age were shaved and anesthetized asdescribed above for the “immunization procedure”. On the day ofimmunization the backs of the mice were wiped with isopropanol. Afterthe alcohol had evaporated (approximately 5 minutes), 100 μl ofphosphate buffered saline (PBS) containing 100 μg of immunostimulatoryDNA (CpG1) and/or cholera toxin (CT) 100 μg was applied to the back with100 μg of a soluble leishmanial antigen extract (SLA) for 90 to 120minutes. SLA is an antigen extract prepared at Walter Reed ArmyInstitute of Research by centrifugal isolation of the soluble proteinsin a sonicate of Leishmazia major promastigotes) extract for 90 to 120minutes. Removal of excess antigen was conducted as described in the“immunization procedure.” The immunization was repeated 4 and 8 weekslater. Twelve weeks after the primary immunization draining (inguinal)LNs were removed and pooled from two immunized animals. The capacity toproliferate in response to media or antigen (SLA) was assessed in astandard 4 day proliferation assay using 3-H incorporation as a readout.The results are shown in Table 11.

Co-administration of SLA and CpG1 (immunostimulatory DNA containing anunmethylated CpG dinucleotide flanked by two 5′ purines and two 3′pyrimidines—SEQ ID NO:2) or CT resulted in a detectable increase in theantigen specific proliferative response. However, the antigen (SLA)specific proliferative response was approximately 20 times higher inlymph node cell cultures from animals exposed simultaneously to bothCpG1 and CT as compared to cultures derived from animals exposed toeither adjuvant alone. Thus it appears that bacterial DNA containingappropriate motifs synergizes with ADP ribosylating exotoxins such as CTas adjuvants on the skin to induce higher immune responses than toeither adjuvant alone.

TABLE 11 Synergy between immunostimulatory DNA and ADP ribosylatingexotoxin (CT) as adjuvants when applied to the skin proliferation (cpm)3-H incorporation in vitro to antigens substances applied in vivo mediaSLA normal LN 180 219 SLA 200 159 SLA/CpG1 1030 2804 SLA/CT 232 2542SLA/CpG1/CT 2232 47122

Example 12

Transcutaneous immunization induces potent immune responses when used asa method of delivery alone. We also have found that transcutaneousimmunization can be used in sequence with other routes of delivery tostimulate an immune response.

BALB/c mice 6 to 8 weeks of age On day 0 both groups of animals receiveda 50 μl intramuscular (im.) injection of DT (5 μg) mixed with alum(Rehydrogel-25 μg in NaCl) into the hind thigh. Eight and 16 weeks latermice in the im/tc/tc group were shaved, anesthetized and immunized bythe transcutaneous route as described above for the “immunizationprocedure” using 100 μg cholera toxin as adjuvant and 100 μg diphtheriatoxoid as antigen. The immunization solution was applied to the back for90 to 120 minutes. Removal of excess antigen was conducted as describedin the “immunization procedure.” Twenty two weeks after the primaryimmunization the animals were bled and the anti-DT titers determinedusing an ELISA as described above for “ELISA IgG (H+L)”. The results areshown in Table 12.

A single im. injection of 5 μg of DT induced a detectable rise in theserum anti-DT titers as compared with titers in sera collected from thesame animals prior to immunization (prebleed). Boosting of the im.primed animals using the transcutaneous immunzation method resulted inan 60 fold rise in the geometric mean titer and clearly alltranscutaneously boosted animals had higher anti-DT titers that thoseobserved in the im. primed group. Thus transcutaneous immunization canbe used to boost antigen specific titers in mice in which the primaryimmunization with the antigen was by the i.m. route. We have also foundthat im primed animals can be boosted by transcutaneous immunization(TCI). Various combinations of TCI priming or boosting with other routesand schedules can be visualized including oral, buccal, nasal, rectal,vaginal, intradermal, by gun or other means of delivery. Additionally,antigens may differ in route and composition including proteinalternating with glycoprotein, subunit with holotoxin, DNA primingfollowed by protein, nucleic acid by im followed by nucleic acid by TCI.Transcutaneous immunization may be used to boost children primed ininfancy or adults primed in childhood. The ease of delivery may enhancethe efficacy vaccines such as the influenza vaccines by allowingmultiple boosts using a patch.

TABLE 12 Boosting of im. primed animals using the transcutaneousimmunization method Anti-DT IgG (H + L) ELISA units Route of Animal #adjuvant/antigen administration prebleed week 22 8563 DT im. 54227 8564DT im. 11833 8565 DT im. 106970 8566 DT im. 10830 8567 DT im. 4003geometric 19711 mean pooled 20 prebleed 8568 DT/ct + dt/ct + dtim./tc./tc. 628838 8569 DT/ct + dt/ct + dt im./tc./tc. 2035507 8570DT/ct + dt/ct + dt im./tc./tc. 1164425 8571 DT/ct + dt/ct + dtim./tc./tc. not available 8572 DT/ct + dt/ct + dt im./tc./tc. 1263138geometric 1171368 mean pooled 10 prebleed 8558 DT/DT/DT im./im./im. notavailable 8559 DT/DT/DT im./im./im. 542669 8560 DT/DT/DT im./im./im.770150 8561 DT/DT/DT im./im./im. 545894 8562 DT/DT/DT im./im./im. 671898geometric 625721 mean pooled 15 prebleed

Example 13

Because TCI appears to stimulate the immune system in such a potentfashion, it is possible that an adjuvant place on the skin at one sitemay act as an adjuvant for an antigen placed at another site. BALB/cmice 6 to 8 weeks of age were anesthetized and shaved as described inthe “immunization procedure”. Animals were not ear tagged but kept incages labeled A, C or G. On the day of immunization the dorsal surfaceof the mouse ear was treated by gently rubbing the outer skin surfacewith a cotton-tipped applicator containing 70% isopropanol. After 5minutes the excess water was blotted from water-treated ears andadjuvant (CT 50 μg) and/or antigen (100 μg of bovine serum albumin (BSA)was applied to the left or right ear surface (described in table) in 50μl phosphate buffered saline. At two and a half hours, the ears wererinsed and blotted dry twice. Mice were boosted in a similar fashionfour and eight weeks later. Twelve weeks after the primary immunizationthe animals were bled and the anti-BSA titers determined using an ELISAas described above for “ELISA IgG (H+L)”. The results are shown in Table13.

Application of BSA alone to the skin was poorly immunogenic with only 1of 5 animals developing an ELISA titer above 100 EU. In contrast, 9 of 9animals receiving CT and BSA on the skin developed titers above 100 EU.Of the animals receiving antigen and adjuvant, the mice given thematerials at the same site (left ear) developed higher (10 fold)anti-BSA titers than animals receiving antigen and adjuvant in separate(left and right) ears. However, animals receiving antigen on one ear andadjuvant on another ear developed an anti-BSA immune response that wasapproximately 30 times higher that animals given BSA alone. Thus,antigen and adjuvant may be topically applied during TCI at differentsites to elicit a humoral immune response. This immunostimulation may beexpected to occur with antigen delivered by other routes and schedulescan be visualized including oral, buccal, nasal, rectal, vaginal,intradermal, by gun or other means of delivery. Additionally, adjuvantsmay be used with nucleic acid immunization to enhance the response. Sucha delivery may not need to be simultaneous to enhance the immuneresponse. For example, an im injection of plasmid DNA may be followedlater by transcutaneous administration of adjuvant. Immunostimulation byCT, LT, TNF∝, CpGs or similar adjuvants is a surprising result becauseit has been thought that molecules 500 daltons could not pass throughthe skin.

TABLE 13 Delivery of antigen and adjuvant at the same or distal sites onthe skin with penetration enhancement. Anti-BSA IgG (H + L) ELISA unitsAnimal # adjuvant/antigen prebleed week 12 group G BSA left ear 240group G BSA left ear 99 group G BSA left ear 40 group G BSA left ear notavailable group G BSA left ear 15 Geometric mean 61 pooled prebleed 6group C CT/BSA left ear 16418 group C CT/BSA left ear 24357 group CCT/BSA left ear 13949 group C CT/BSA left ear 70622 group C CT/BSA leftear not available Geometric mean 25053 pooled prebleed 3 group A CTleft/BSA right ear 106 group A CT left/BSA right ear 23806 group A CTleft/BSA right ear 1038 group A CT left/BSA right ear 1163 group A CTleft/BSA right ear 8696 Geometric mean 1939 pooled prebleed 15

Example 14

Transcutaneous Immunization in Humans

The invention can be practiced using a suitable vehicle or carrier. Forexample a patch can be used as such a vehicle and can be treated withthe formulation of the invention or can be used to cover the area of theskin which has been treated with the formulation of the invention. Asuitable patch can be fabricated from, for example, cotton, nylon,rayon, polyester or combinations thereof. Such patches can be providedwith an adhesive or non-adhesive backing. Patches with a non-adhesivebacking can be secured to the animal by non-adhesive means, such as, forexample, wrapping. Suitable backing can be fabricated from materialssuch as, for example, silicon, acrylate or rubber. Other vehicles orcarriers which can be used include those listed above; such as forexample, powders, oils, water, cream and the like. To evaluate thepotential for TCI in humans, a Phase I trial was conducted using LT toattempt to induce serum anti-LT antibodies. Six volunteers received adose of 500 μg of LT, a dose similar to oral adjuvant doses used for acholera vaccine (1 mg CTB). LT was produced under GMP conditions at theSwiss Serum and Vaccine Institute (Berne, Switzerland) and was providedby Oravax Inc., Cambridge, Mass. The volunteers received 500 μg of LTmixed in 500 μl of sterile saline which was absorbed on a 2 in² cottongauze pad with polyvinyl backing and covered by a 4×4 in²Tegaderm™dressing. The immunization was conducted by placing the patchon unmanipulated skin for 6 hours after which the site was thoroughlyrinsed with 500 ml of sterile saline. Individuals were similarlyreimmunized after 12 weeks. Volunteers were examined on days 1, 2, 3 and7 for signs of inflammation at the site of immunization and interviewedfor symptoms related to the vaccination. The immunization was conductedby placing the patch on unmanipulated skin for 6 hours after which thepatch was removed and the site was thoroughly rinsed with saline.Individuals were reimmunized after 12 weeks. No adverse reactions wereseen, either systemically or at the site of immunization after the firstor second immunization. Anti-LT IgG titers were determined as previouslydescribed. Results are reported in ELISA units (EU) which are defined asthe inverse dilution of sample that yields an OD of 1.0. Anti-LT IgA wasdetermined in the same manner as anti-LT IgG using goat anti-humanIgA(α)-HRP (Kirkegaard and Perry, Gaithersburg, Md.) against a standardIgA curve made using human IgA (ICN). As shown in Table 14, 6 of 6individuals responded by producing a rise in serum anti-LT IgG or IgAantibodies, defined as a four-fold increase in antibody titers. The meanfold rise in anti-LT IgG was 10.2 and the mean fold rise in serumanti-LT IgA was 7.2. Biopsies of the immunization site and contralateralarm showed no signs of inflammation of the skin. These results confirmthat TCI can be conducted in humans without skin irritation orinflammation.

Suitable patch materials have been previously described. In general thepatch may consist of, for example, a pressure sensitive dressing, amatrix for or absorbant layer to carry the vaccines and adjuvant, avaccine impermeable backing and a release liner. This and other suitablepatch examples are described in U.S. Pat. Nos. 4,915,950 and 3,734,097.

Patches can be fabricated to include woven and non-woven matrices ofmaterials to include, for example, polyester/cellulose, polypropylene,polyester, polyester/rayon and the like.

Examples of non-woven patch matrices can include:

BBA Nonwovens

-   -   a. Grade# 1313290, wet laid non-woven,        composition=polyester/cellulose, weight (gsy)=35.4, weight        (gsm)=42.3, thickness (mils)=7.9, tensile MD=3.4, tensile        CD=2.4.    -   b. Grade# 2006086, thermal bond non-woven,        composition=polypropylene, weight (gsy)=16.0, weight (gsm)=19.1,        thickness (mils)=10.2, tensile MD=3.3, tensile CD=0.7.    -   c. Grade# 149146, thermal bond non-woven, composition=polyester,        weight (gsy)=25.6, weight (gsm)=30.6, thickness (mils)=6.5,        tensile MD=5.3, tensile CD=0.9.    -   d. Grade# 149020, thermal bond non-woven,        composition=polyester/rayon, weight (gsy)=30.5, weight        (gsm)=36.4, thickness (mils)=13.2, tensile MD=5.5, tensile        CD=1.1.    -   e. Grade# 140-027, hydroentangled nonwoven,        composition=polyester/rayon, weight (gsy)=28.0, weight        (gsm)=33.5, thickness (mils)=22.4, tensile MD=10.4, tensile        CD=3.8.

Pressure sensitive adhesive that can be used in the present inventioninclude, for example adhesive based on acrylate, silicone, rubber andthe like.

TABLE 14 Mean fold rise in human anti-LT IgG and IgA Volunteer # 4 weekIgG 12 week IgG 16 week IgG 13 15.2 9.5 12.5 14 1.4 1.6 1.7 15 11.7 15.012.9 16 1.3 0.7 16.0 17 12.5 51.9 58.6 18 1.3 2.1 4.3 Mean rise IgG 4.25.0 10.2 Volunteer # 4 week IgA 12 week IgA 16 week IgA 13 7.2 4.1 10.114 4.9 4.3 4.3 15 4.9 5.7 4.5 16 1.4 1.3 7.0 17 15.3 29.4 28.1 18 1.31.5 3.5 Mean rise IgA 4.1 4.2 7.2

Example 15

LT has been shown to be effective for immunizing humans by thetranscutaneous route. We also have found that LT acts as an adjuvant forTCI. C57BL/6 mice 6 to 8 weeks of age were anesthetized and shaved asdescribed in the “immunization procedure”. On the day of immunizationthe backs of the mice were wiped with isopropanol. After the alcohol hadevaporated (approximately 5 minutes), 100 μl of phosphate bufferedsaline (PBS) containing 100 μg of heat labile enterotoxin (LT) and/or100 μg of diphtheria toxoid (DT) was applied to the back for 90 to 120minutes. Removal of excess antigen was conducted as described in the“immunization procedure.” The immunization was repeated 4 and 8 weekslater. Ten weeks after the primary immunization the animals were bledand the anti-DT titers determined using an ELISA as described above for“ELISA IgG (H+L)”. The results are shown in Table 15.

Anti-DT titers were clearly elevated in serum from animals immunizedwith LT and DT when compared with titers in serum from animals treatedwith DT alone or those in prebleed serum samples. Thus it appears thatheat labile enterotoxin (LT) can be used as an adjuvant on the skin.

TABLE 15 Use of heat labile enterotoxin (LT) from E. coli as an adjuvanton the skin. Anti-DT IgG (H + L) ELISA units Animal # adjuvant/antigenprebleed week 10 51 DT 11 52 DT 7 53 DT 4 54 DT 8 55 DT 7 geometric mean7 pooled prebleed 4 71 LT/DT 7126 72 LT/DT 46909 73 LT/DT 669 74 LT/DT8480 75 LT/DT 1598 geometric mean 4970 pooled prebleed 5

Example 16

To assess the role of physical/mechanical penetration enhancement, thesuperficial layers of the stratum corneum were removed by tapestripping. Tape stripping is an intervention well known in the art toremove the outer layer of the stratum corneum. C57BL/6 mice 6 to 8 weeksof age were anesthetized and shaved as described in the “immunizationprocedure.” Twenty four hours later, CT (25 μg) was applied to the backsof the mice in 50 μl of phosphate buffered saline; “none” interventiongroup. Alternatively, the skin on the backs of a second group of animalswas subjected to mild tape stripping; “tape stripping” interventiongroup. The tape stripping procedure was accomplished by applyingcellophane scotch-tape to the backs, allowing bonding to the skinsurface for 3 minutes, followed by gentle removing of the tape. Thebonding/removal steps were repeated 3 times. CT (25 μg) was then appliedto the backs of the mice in 50 μl of phosphate buffered saline. Antigenremained on the backs for approximately 1.5 hrs at which time removal ofexcess antigen was conducted as described in the “immunizationprocedure.”

The anti-CT antibody titers were determined using ELISA as describedabove for “ELISA IgG (H+L)” using sera collected 11 days after theprimary immunization. The results are shown in Table 16. CT wasimmunogenic in both groups as compared to sera collected from the sameanimals prior to immunization (prebleed). However, the geometric meantiter in the tape stripped group was 100 fold higher and the titers weremore consistent among the latter animals as compared with the “none”animals. Thus it appears that physical disruption of the skin surfaceusing tape stripping enhances delivery of antigen by the transcutaneousroute.

This simple device could be replaced by other physical penetrationdevices to deliver antigens and adjuvants into the epidermis such as aneedle and tuberculin syringe used for intradermal injection,microneedles that are of length to penetrate only the stratum corneum orsuperficial dermis, devices used for TB tine testing, gas powered guns,adhesive tape for tape stripping, or other devices known to penetrateonly into the epidermis or superficial dermis. Tape stripping devicescould be used in conjunction with other penetration enhancers. Tapestripping devices may be used in conjunction with a marker to delineatethe site for patch placement, and may be dispersed in a roll or inindividual units.

TABLE 16 Enhancement of transcutaneous immunization by physicalpenetration enhancement: Anti-CT titers in mice that had the skinstripped using cellophane tape before application of the antigen.anti-CT IgG (H + L) Elisa units Animal # intervention prebleed day 11976 none 155 977 none 4 978 none 4 979 none 31 980 none 23 geomean 16prebleed 2 986 tape strip 10702 987 tape strip 1285 988 tape strip 5832989 tape strip 997 990 tape strip 782 geomean 2990 prebleed 3

Example 17

Nucleic acids such as plasmid DNA or RNA can be used to induce andimmune response and are well known in the art. The use of Nucleic acidsin transcutaneous immunization was described in previous patents(PCT/US97/21324). The use of nucleic acids (also known as geneticimmunization) on the skin with penetration enhancement techniques isillustrated in the following example.

C57BL/6 mice 6 to 8 weeks of age were anesthetized and shaved asdescribed in the “immunization procedure”. For the “NP DNA” group themice were wiped with isopropanol, after the alcohol had evaporated(approximately 10 minutes), the backs were hydrated with water using asaturated gauze pad. Approximately 10 minutes later the any excess waterwas blotted off and a 100 μg of a DNA plasmid (pCMV-NP) encoding forinfluenza nucloprotein was applied to the back in 100 μl of saline. Asecond group of NP-DNA mice were subjected to the same immunizationprotocol except that their backs were tape stripped 3× prior to alcoholswabbing; “NP DNA—tape stripping”. The tape stripping procedure wasaccomplished by applying cellophane scotch-tape to the backs, allowingbonding to the skin surface for 5 minutes, followed by gentle removingof the tape. A third group of mice was engaged in the tapestripping/immunization protocol described and 100 μg of the adjuvantheat labile enterotoxin (LT) was included in the immunization solution.Sixteen days after the primary immunization the animals were bled andthe anti-influenza NP titers determined using an ELISA as describedabove for “ELISA IgG (H+L)”. The results are shown in Table 17.

Anti-influenza titers were determined using a split virus antigen(Fluzone) preparation to coat the ELISA plates. ELISA titers weredetermined in 5 individual animals and the mean optical density readingfor each group is shown. All three immunization groups developedanti-influenza titers as compared with titers in serum collected fromthe same animals prior to immunization (prebleed). As compared with theNP DNA alone group, tape stripping prior to immunization enhanced theanti-Influenza titer in all three serum dilutions tested (1:100, 1:200,1:400) and addition of an adjuvant (LT) further enhanced this response.Thus, DNA can be used on the skin to induce immune responses to vaccineantigens and its effectiveness can be enhanced by the addition ofadjuvants and penetration enhancement such as tape stripping.

TABLE 17 Immuogenicity of DNA applied as antigen on the skin usingalcohol penetration enhancement. Anti-INF IgG optical density (405 nm)day 16 prebleed post immunization Antigen/adjuvant intervention 1:1001:100 1:200 1:400 NP DNA none 0.21 0.47 0.20 0.07 NP DNA tape stripping0.39 0.64 0.28 0.13 NP DNA/LT tape stripping 0.39 0.87 0.38 0.13

Example 18

Transcutaneous immunization (TCI), because of the ease of delivery,allows the application to be given over different draining lymph nodes.This may have an additional advantage in that it enhances the immuneresponse. Rabbits were anesthetized, shaved, and immunized as describedabove. Animals were immunized with 100 μg cholera toxin (CT) and 100 μgof influenza hemagglutinin (HA) at one site or two sites on the back. HAand CT were applied at 0, 3 and 5 weeks. Seven weeks after the primaryimmunization, the animals were bled and the anti-HA titers determinedusing an ELISA as described above for “ELISA IgG (H+L)”. The results areshown in Table 18.

Anti-HA titers were elevated in serum from 10 of 10 animals immunizedwith CT and HA when compared with titers in serum from the same animalsprior to immunization (prebleed). The geometric mean titer in the twosite group was 3 fold higher than that in the one site group suggestingthat antigen delivery at multiple sites may be used to enhance TCI.Thus, antigens can be delivered by TCI either at a single or multiplesites on the skin.

TABLE 18 Transcutaneous delivery of antigen in a single or multiplesites. anti-HA IgG (ELISA units) Animal antigen/adjuvant prebleed 7weeks geomean 1 CT/HA one site <25 1142 2596 2 CT/HA one site <25 9617 3CT/HA one site <25 2523 4 CT/HA one site <25 2275 5 CT/HA one site <251869 6 CT/HA two sites <25 10348 8403 7 CT/HA two sites <25 18453 8CT/HA two sites <25 9778 9 CT/HA two sites <25 15985 10 CT/HA two sites<25 1404

Example 19

The variety of antigens which can be delivered by TCI is furtherillustrated by the use of a polysaccharide conjugate vaccine to induceanti-polysaccharide antibodies. BALB/c mice 6 to 8 weeks of age wereanesthetized, shaved, and immunized as described in the “immunizationprocedure”. Mice were immunized with cholera toxin (CT) and Haemophilusinfluenzae B polysaccharide (Hib-PS) at 0, 3 and 5 weeks. Seven weeksafter the primary immunization, the animals were bled and theanti-Hib-PS titers determined using an ELISA as described above for“ELISA IgG (H+L)”. The results are shown in Table 19.

Anti-Hib-PS titers were elevated in serum from 4 of 10 animals immunizedwith CT and Hib-PS when compared with titers in serum from the sameanimals prior to immunization (prebleed). Thus TCI can be used to induceanti-polysaccharide antigens through the skin. This is a common humanuse vaccine antigen and represents and important strategy forimmunization.

TABLE 19 Delivery of a conjugated polysaccharide by transcutaneousimmunization anti-Hib PS IgG (μg/ml) ear tag # antigen/adjuvant prebleed7 weeks 1 CT/Hib-PS (100 μg/100 μg) <0.20 <0.20 2 CT/Hib-PS (100 μg/100μg) <0.20 <0.20 3 CT/Hib-PS (100 μg/100 μg) <0.20    1.68 4 CT/Hib-PS(100 μg/100 μg) <0.20 <0.20 5 CT/Hib-PS (100 μg/100 μg) <0.20    1.86 6CT/Hib-PS (100 μg/25 μg) <0.20    1.04 7 CT/Hib-PS (100 μg/25 μg) <0.20<0.20 8 CT/Hib-PS (100 μg/25 μg) <0.20 <0.20 9 CT/Hib-PS (100 μg/25 μg)<0.20    6.30 10 CT/Hib-PS (100 μg/25 μg) <0.20 <0.20

Example 20

Transcutaneous immunization of mice with human use vaccine antigens CThas been shown to act as adjuvant for transcutaneous immunization withsingle toxoids and BSA. We transcutaneously immunized mice with avariety of human-use vaccine antigens, including a multivalent toxoidvaccine (tetanus and diphtheria toxoids), a yeast expressed recombinantprotein(HIV p55 gag) and whole killed rabies viruses using CT as anadjuvant as shown in Table 20. BALB/c mice (n=5) were immunized andboosted twice as previously described (Glenn, G. M., Scharton-Kersten,T., Vassell, R., Matyas, G. & Alving. C. R. Transcutaneous immunizationusing bacterial ADP-ribosylating exotoxins as antigens and adjuvants.Infect. Immun. (in the press). Immunizing doses included 100/50/50 μgCT/TT/DT via TCI versus 3/1/1 for alum/TT/DT IM; 100/100 μg for LT+DTversus 100 μg of DT alone. 100/100 μg for CT/p55 via TCI versus 100 μgp55 alone. Mice immunized with 17 IE of killed rabies virus (n=10) hadbeen primed intramuscularly 2× and then boosted transcutaneously (17 IE)after light alcohol swabbing of the skin and compared to 3×IM rabiesimmunization. Antibody levels against DT, TT, p55 and rabies weredetermined using ELISA as previously described (Grassi, M., Wandler, A.& Peterhans, E. Enzyme-linked immunoabsorbant assay for determination ofantibodies to the envelope glycoprotein of rabies virus. J. Clin.Microbiol. 27, 899-902 (1989). Miyamura, K., Tajiri, E., Ito, A.,Murata, R. & Kono, R. Micro cell culture method for determination ofdiphtheria toxin and antitoxin titres using VERO cells. II. Comparisonwith the rabbit skin method and practical application forseroepidemiological studies. J. Biol. Stand. 2, 203-209 (1974)). TCIresulted in similar increases in the antibody responses to TT and DT andthe anti-DT neutralization titers were comparable to that elicited byintramuscular immunization (IM). These data show that TCI may be used toinduce immune response of comparable magnitude as those induced byexisting immunization practices. TCI boosting of IM primed animals alsoresulted in a significant rise in anti-rabies titers in all 10 animalstested (0.53 to 1.03 IU, p<0.02, Student's t-test). Antibodies to theantigens DT and p55 administered without adjuvants were very low orundetectable, consistent with our previous observations that antigensare only weakly immunogenic when applied without adjuvant. LT also actedas adjuvant (Table 20) in a fashion similar to previous studies using CT(1,2). Although the immunizations were not optimized as compared tointramuscular delivery, these antigen-specific responses confirm thatTCI may be used for a variety of human-use vaccines from a variety ofsources an a variety of sizes and that LT can act as an adjuvant forcoadministered vaccine antigens.

TABLE 20 Murine antibody responses to human-use vaccine antigensadministered by TCI Immunizing Anti- Antibody TCI IM/alum gen(s) for TCIspecificity (ELISA Units) (ELISA Units) CT + TT + DT Anti-DT 135,79285,493 (86,552-146-759) (24,675-238,904) CT + TT + DT Anti-TT 30,05194,544 (13,863-53,174) (74,928-113,408) CT + TT + DT Diphtheria 4041,226 toxin (22-2816) (352-11,264) neutralization LT + DT Anti-DT 4976ND (669-46,909) CT + HIV p55 Anti-p55 10,630 ND gag (1063-52,597) CT +Killed Anti-G protein 1.03 (IU/ml) 7.54 (IU/ml) Rabies Virus (0.31-2.77)(3.31-17.47) ND = not done. ELISA units (EU) shown as geometric mean andrange in brackets.

Example 21

Langerhans Cell Activation

In two subjects, the site of immunization and the contralateralunimmunized arm were biopsied, one at 24 hours post-immunization and oneat 48 hours after the second immunization. Hematoxylin and eosin (H&E)staining of the specimens confirmed the clinical findings suggestingthat no inflammation was seen after the immunization (FIG. 1 A,B).Although routine histologic sections were unremarkable, LCs visualizedusing anti-CD1 a staining of specimens from the site of immunizationdemonstrated greatly enlarged cell bodies but otherwise normal numbersof cells when compared to the control biopsies from the opposite arm,both at 24 and 48 hours (FIG. 1 C,D,E,F). Similar findings were seenusing anti-HLA-DR and anti-S-100 to visualize LCs (not shown). LCmorphology in the TCI immunized skin was similar in appearance totonsilar crypt LCs that are thought to be chronically activated bylipopolysaccharides from the flora of the mouth (Noble).

Because of the limited size and number of human skin biopsy specimensexamined, complementary murine studies were performed. LC activation inmurine systems using contact sensitizers, LPS, and proinflammatorycytokines is characterized by both changes in morphology (Aiba) andthrough elevations in surface marker expression (Jakob and Udey). Mouseear skin is frequently used for studies of LC activation and has alsobeen shown to be an excellent site for transcutaneous immunization(Scharton-Kersten). Epidermal sheets were prepared 24 hours afterapplication of CT to the ear and stained for MHC class II, anLC-restricted marker in murine skin. In comparison to the PBS treatedears, FIG. 2A, LC in CT treated ears exhibited marked changes in LCmorphology with loss of dendritic processes, enlarged cell bodies, andintense staining of the cells—features of LC activation (Aiba) (FIG. 2B,C). The LC-activating potential of CT was confirmed using flowcytometry. LC from CT-treated skin expressed increased levels of MHCClass II antigens and CD 86 (B7-2) and decreased levels of E-cadherin,consistent with LC activation described elsewhere (Pierre, Aiba, Jakob).

Immunization Procedure which May be Used for Example 22.

BALB/c mice may be shaved with a #40 clipper. This shaving could be donewithout any signs of trauma to the skin. The shaving could be done fromthe mid-thorax to just below the nape of the neck. The mice can then beallowed to rest for 24 hours. Prior to this the mice could be ear-taggedfor identification, and pre-bled to obtain a sample of pre-immune serum.Mice could also be transcutaneously immunized without shaving byapplying 5-500 μl of immunizing solution to each ear. The mice could beimmunized in the following way. Mice could be anesthetized with0.03-0.06 ml of a 20 mg/ml solution of xylazine and 0.5 ml of 100 mg/mlketamine and immobilized by this dose of anesthesia for approximately1-3 hours. The mice could be placed ventral side down on a warmingblanket.

The immunizing solution and penetration enhancement compound (ortechnique) could be placed on the dorsal shaved skin of a mouse in thefollowing manner: a 1.2 cm×1.6 cm stencil made of polystyrene is laidgently on the back and a saline-wetted sterile gauze could be used topartially wet the skin (allowing even application of the immunizingsolution), the immunizing solution could then be applied with a pipet tothe area circumscribed by the stencil to yield a 2 cm² patch ofimmunizing solution. Care could be used not to scrape or rub the skinwith the pipet tip. The immunizing solution could be spread around thearea to be covered with the smooth side of the pipet tip. Alternatively,the immunizing solution could be place directly on the skin withoutwetting or with wetting without the use of a stencil.

The immunizing solution (between about 5 μl and about 200 μl) could beleft on the back of the mouse for 60-120 minutes. At the end of theimmunization period, the mouse could be held gently by the nape of theneck and the tail under a copious stream of lukewarm tap water, andwashed for 10 seconds. The mouse could then be gently patted dry with apiece of sterile gauze and a second washing could then be performed for10 seconds; the mouse could then be patted dry a second time and left inthe cage. The mice would appear to exhibit no adverse effects from theanesthesia, immunization, washing procedure, or toxicity from theexotoxins. No skin irritation, swelling or redness would be seen afterthe immunization and the mice would appear to thrive. Immunization usingthe ear could be performed as described above except that fur would notbe removed prior to immunization.

Antigen

The following antigens could be used for immunization and ELISA, andcould be mixed using sterile PBS or normal saline. Cholera toxin or CT(List Biologicals, Cat #101B, lot #10149CB), CT B subunit (ListBiologicals, Cat #BTO1, lot #CVXG-14E), CT A subunit (List Biologicals,Cat #102A, lot #CVXA-17B), CT A subunit (Calbiochem, Cat #608562);pertussis toxin, salt-free (List Biologicals, lot #181120a); tetanustoxoid (List Biologicals, lots #1913a and #1915a); Pseudomonas exotoxinA (List Biologicals, lot #ETA25a); diphtheria toxoid (List Biologicals,lot #15151); heat-labile enterotoxin from E. coli (Sigma, lot #9640625);bovine serum albumin or BSA (Sigma, Cat #3A-4503, lot #31F-0116); andHemophilus influenza B conjugate (Connaught, lot#6J81401).

ELISA—IgG(H+L)

Antibodies specific for CT, LT, ETA, pertussis toxin, diphtheria toxoid,tetanus toxoid, Hemophilus influenza B conjugate, and BSA could bedetermined using ELISA in a technique similar to Glenn et al. (1995).

Example 22

Activation of LT may be performed by incubating the LT with trypsin (ortrypsin immobilized on beads) with or without reducing agents (e.g.,dithiotheritol) to break the disulphide bonds near the trypsin cleavagesite, under standard reaction conditions. Native LT can be activated byincubation of 100 μg of protein with 0.1 μg trypsin in a total reactionvolume of 100 μl for 45 min at 37° C. Alternatively, the trypsin can befixed to beads and the LT may be eluted over the trypsin beads. Trypsincleavage can be demonstrated by SDS-PAGE (Laemilli, U.K., 1970, Cleavageof structural proteins during the assembly of the head of bacteriophageT4, Nature 227:680-685). LT either treated or not treated with trypsincan be mixed with buffer containing dithiothreitol, and heated to 100°C. for 5 min prior to SDS-PAGE analysis. Trypsin-treated LT could have aproteolytic fragment of 21K daltons consistent with trypsin cleavage ofthe A1 and A2, allowing the A1 subunit to ADP-ribosylate G proteins andtherefore exert its toxic effects whereas untreated LT would demonstratea band at 28K daltons, consistent with an intact A subunit. Activationcan further be demonstrated in the mouse Y-1 cell assay in which nativeLT would be 1,000 fold less active than CT, but trypsin-treated LT wouldbe equally as active as CT. Activation can also be demonstrated using anenzymatic assay, the NAD:agmatine ADP-ribosyltransferase assay. In suchan assay, non-trypsin-treated LT would be expected to show low orundetectable activity whereas trypsin-treated LT would be expected toshow similar activity as that demonstrated by CT.

Example 23

Transcutaneous immunization may be more useful if the immunization canbe performed over a short period of time. It may be useful for examplefor an immunization to be performed during a routine clinic visitlasting 30 minutes. In this example we show that transcutaneousimmunization can be performed in hydrated, alcohol swabbed skin in sucha short period.

C57BL/6 mice 6 to 8 weeks of age were anesthetized and shaved asdescribed in the “immunization procedure”. On the day of immunizationthe backs of the mice were wiped with isopropanol. After the alcohol hadevaporated (approximately 10 minutes), 200 μl of water was applied tothe back for hydration. 15 minutes later the immunization solution wasapplied to the back and left for the specified period of time. Removalof excess antigen was conducted as described in the “immunizationprocedure.” Mice were immunized with CT alone (100 μg in 50 μl) at dOand with CT plus DT (100 μg each in 100 VI volume) at 4, 6 and 9 weeks.Twelve weeks after the primary immunization the animals were bled andthe anti-DT titers determined using an ELISA as described above for“ELISA IgG (H+L)”. The results are shown in Table 23.

Anti-DT titers were clearly elevated in serum from all of the animalsimmunized with CT and DT when compared with titers in serum from thesame animals prior to immunization (prebleed). Maximal effects ofimmunization appeared to occur in animals vaccinated for a period of 60minutes although the titers were similar at 30 and 120 minutes. Fifteenminutes of immunization seemed less efficient as the titers in thisgroup were approximately 10 fold less than that observed in the 30, 60and 120 minute groups. Thus it appears that TCI can be achieved within15 minutes of antigen application.

TABLE 23 Effect of the duration of antigen application on humoralimmunity induced by transcutaneous immunization. duration of anti-DT IgG(ELISA units) ear tag # immunization prebleed 12 weeks geomean 361 15min 6 214 300 362 15 min 664 363 15 min 314 364 15 min 181 365 15 min1594 366 30 min 8 11953 13445 367 30 min 32478 368 30 min 24346 369 30min 3457 370 30 min 99776 371 60 min 12 75787 107963 372 60 min 200768373 60 min 102592 374 60 min 87034 375 60 min 9210 376 120 min 4 4813248202 377 120 min 99362 378 120 min 37308 379 120 min 30255 380 120 min25149

All publications referred to in this application are incorporated byreference herein as indicative of the state of the art.

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1. A method for inducing an antigen-specific immune response in asubject comprising: a) pretreating an area of the skin of the subject,wherein pretreating comprises applying means for enhancing penetrationand/or barrier disruption of the skin; and b) applying a formulationtranscutaneously to the pretreated area to induce an antigen-specificimmune response, wherein the formulation comprises: 1) an antigen in anamount effective to induce an antigen-specific immune response; 2) anadjuvant present in an amount effective to enhance the immune responseto the antigen; and, 3) a pharmaceutically acceptable carrier; whereinpretreating enhances the immune response.
 2. The method of claim 1,wherein pretreating comprises applying to the skin a chemical means, aphysical means, a mechanical means, a hydration means, or a combinationthereof.
 3. The method of claim 1, wherein pretreating comprisesapplying a chemical to the skin.
 4. The method of claim 3, wherein thechemical is an alcohol, an acetone, a detergent, a depilatory agent, akeratinolytic formulation, a cream, or a combination thereof.
 5. Themethod of claim 1, wherein pretreating comprises applying a device. 6.The method of claim 5, wherein the device is selected from the groupconsisting of a propellant device, a device comprising tines, a devicecomprising microneedles, a device comprising a tine disk, a tapestripping device, a gas powered gun, a swab, an emery board, an abrasivepad, an electroporation device, an ultrasound device, and aniontophoresis device.
 7. The method of claim 1, wherein the antigen is anucleic acid encoding an antigen, carbohydrate, a glycolipid, aglycoprotein, a lipid, a lipoprotein, phospholipid, a polypeptide, aprotein, a fusion protein, or chemical conjugate of a combinationthereof.
 8. The method of claim 1, wherein the antigen is derived from apathogen.
 9. The method of claim 8, wherein the pathogen is a virus, abacterium, a parasite, or a fungus.
 10. The method of claim 9, whereinthe virus is an influenza virus or a rabies virus.
 11. The method ofclaim 10, wherein the antigen is hemagglutinin A.
 12. The method ofclaim 9, wherein the bacterium is E. coli or Bacillus anthracis.
 13. Themethod of claim 12, wherein the antigen is E. coli heat-labileenterotoxin (LT).
 14. The method of claim 12, wherein the antigen is anucleic acid encoding E. coli heat-labile enterotoxin (LT).
 15. Themethod of claim 1, wherein the antigen is a pathogen.
 16. The method ofclaim 15, wherein the pathogen is a virus, a bacterium, a parasite, or afungus.
 17. The method of claim 16, wherein the virus is a whole virus,a live virus, an attenuated live virus, an inactivated virus, adetergent treated virus, or a combination thereof.
 18. The method ofclaim 17, wherein the virus is an influenza virus or a rabies virus. 19.The method of claim 18, wherein the influenza virus compriseshemagglutinin A.
 20. The method of claim 16, wherein the bacterium is Ecoli or Bacillus anthracis.
 21. The method of claim 20, wherein the E.coli comprises E. coli heat-labile enterotoxin (LT).
 22. The method ofclaim 1, wherein the antigen is a multivalent antigen.
 23. The method ofclaim 1, wherein the adjuvant comprises a molecule selected from thegroup consisting of a bacterial ADP-ribosylating exotoxin (bARE), abinding B subunit of a bARE, a toxoid of a bARE, a genetically alteredbARE, and a genetically detoxified mutant of a bARE.
 24. The method ofclaim 23, wherein the antigen is an influenza antigen.
 25. The method ofclaim 1, wherein the adjuvant is a nucleic acid encoding a moleculeselected from the group consisting of a bacterial ADP-ribosylatingexotoxin (bARE), a binding B subunit of a bARE, a toxoid of a bARE, agenetically altered bARE, and a genetically detoxified mutant of a bARE.26. The method of claim 25, wherein the nucleic acid encodes E. coliheat labile enterotoxin (LT).
 27. The method of claim 1, wherein theantigen and the adjuvant are the same molecule.
 28. The method of claim27, wherein the molecule is E. coli heat-labile enterotoxin (LT). 29.The method of claim 27, wherein the molecule is hemagglutinin A.
 30. Themethod of 1, wherein the formulation is applied using a patch.
 31. Themethod of claim 1, wherein the adjuvant is selected from the groupconsisting of nucleic acid encoding an adjuvant, bacterial exotoxin,cytokine, chemokine, lipopolysaccharide, a molecule containingunmethylated CpG motifs, a heat shock protein, a derivative of a heatshock protein, tumor necrosis factor, genetically detoxified toxin, andcombinations thereof.
 32. The method of claim 1, wherein the adjuvant isprovided as a nucleic acid comprising a sequence encoding the adjuvant.33. The method of claim 1, wherein the antigen or adjuvant activates anantigen presenting cell.
 34. The method of claim 33, wherein the antigenpresenting cell is a Langerhans cell or a dermal dendritic cell.
 35. Themethod of claim 1, wherein the antigen is a whole microorganism, a wholecell, or a virion.
 36. A method for inducing an antigen-specific immuneresponse in a subject comprising concurrently, a) treating an area ofthe skin of the subject, wherein treating comprises applying means forenhancing penetration and/or barrier disruption of the skin; and b)applying a formulation transcutaneously to the treated area to induce anantigen-specific immune response, wherein the formulation comprises: 1)an antigen in an amount effective to induce an antigen-specific immuneresponse; 2) an adjuvant present in an amount effective to enhance theimmune response to the antigen; and, 3) a pharmaceutically acceptablecarrier; wherein treating enhances the immune response.
 37. The methodof claim 36, wherein treating comprises applying to the skin a chemicalmeans, a physical means, a mechanical means, a hydration means, or acombination thereof.
 38. The method of claim 36, wherein treatingcomprises applying a chemical to the skin.
 39. The method of claim 38,wherein the chemical is an alcohol, an acetone, a detergent, adepilatory agent, a keratinolytic formulation, a cream, or a combinationthereof.
 40. The method of claim 36, wherein treating comprises applyinga device.
 41. The method of claim 40, wherein the device is selectedfrom the group consisting of a propellant device, a device comprisingtines, a device comprising microneedles, a device comprising a tinedisk, a tape stripping device, a gas powered gun, a swab, an emeryboard, an abrasive pad, an electroporation device, an ultrasound device,and an iontophoresis device.
 42. The method of claim 36, wherein theantigen is a nucleic acid encoding an antigen, carbohydrate, aglycolipid, a glycoprotein, a lipid, a lipoprotein, phospholipid, apolypeptide, a protein, a fusion protein, or chemical conjugate of acombination thereof.
 43. The method of claim 36, wherein the antigen isderived from a pathogen.
 44. The method of claim 43, wherein thepathogen is a virus, a bacterium, a parasite, or a fungus.
 45. Themethod of claim 44, wherein the virus is an influenza virus or a rabiesvirus.
 46. The method of claim 45, wherein the antigen is hemagglutininA.
 47. The method of claim 44, wherein the bacterium is E. coli orBacillus anthracis.
 48. The method of claim 47, wherein the antigen isE. coli heat-labile enterotoxin (LT).
 49. The method of claim 47,wherein the antigen is a nucleic acid encoding E. coli heat-labileenterotoxin (LT).
 50. The method of claim 34, wherein the antigen is apathogen.
 51. The method of claim 50, wherein the pathogen is a virus, abacterium, a parasite, or a fungus.
 52. The method of claim 51, whereinthe virus is a whole virus, a live virus, an attenuated live virus, aninactivated virus, a detergent treated virus, or a combination thereof.53. The method of claim 52, wherein the virus is an influenza virus or arabies virus.
 54. The method of claim 53, wherein the influenza viruscomprises hemagglutinin A.
 55. The method of claim 51, wherein thebacterium is E coli or Bacillus anthracis.
 56. The method of claim 55,wherein the E. coli comprises E. coli heat-labile enterotoxin (LT). 57.The method of claim 34, wherein the antigen is a multivalent antigen.58. The method of claim 34, wherein the adjuvant comprises a moleculeselected from the group consisting of a bacterial ADP-ribosylatingexotoxin (bARE), a binding B subunit of a bARE, a toxoid of a bARE, agenetically altered bARE, and a genetically detoxified mutant of a bARE.59. The method of claim 58, wherein the antigen is an influenza antigen.60. The method of claim 34, wherein the adjuvant is a nucleic acidencoding a molecule selected from the group consisting of a bacterialADP-ribosylating exotoxin (bARE), a binding B subunit of a bARE, atoxoid of a bARE, a genetically altered bARE, and a geneticallydetoxified mutant of a bARE.
 61. The method of claim 60, wherein thenucleic acid encodes E. coli heat labile enterotoxin (LT).
 62. Themethod of claim 36, wherein the antigen and the adjuvant are the samemolecule.
 63. The method of claim 62, wherein the molecule is E. coliheat-labile enterotoxin (LT).
 64. The method of claim 62, wherien themolecule is hemagglutinin A.
 65. The method of 36, wherein theformulation is applied using a patch.
 66. The method of claim 36,wherein the adjuvant is selected from the group consisting of nucleicacid encoding an adjuvant, bacterial exotoxin, cytokine, chemokine,lipopolysaccharide, a molecule containing unmethylated CpG motifs, aheat shock protein, a derivative of a heat shock protein, tumor necrosisfactor, genetically detoxified toxin, and combinations thereof.
 67. Themethod of claim 36, wherein the adjuvant is provided as a nucleic acidcomprising a sequence encoding the adjuvant.
 68. The method of claim 36,wherein the antigen or adjuvant activates an antigen presenting cell.69. The method of claim 68, wherein the antigen presenting cell is aLangerhans cell or a dermal dendritic cell.
 70. The method of claim 36,wherein the antigen is a whole microorganism, a whole cell, or a virion.71. A method for inducing an antigen-specific immune response in asubject comprising: a) delivering parenterally a first formulationcomprising an antigen to a subject; b) treating an area of the skin ofthe subject, wherein treating comprises applying means for enhancingpenetration and/or barrier disruption of the skin to enhance the immuneresponse; and c) applying transcutaneously a second formulationcomprising an adjuvant to the area of the skin, thereby inducing anantigen-specific immune response.
 72. The method of claim 71, whereintreating comprises applying to the skin a chemical means, a physicalmeans, a mechanical means, a hydration means, or a combination thereof.73. The method of claim 71, wherein treating comprises applying achemical to the area of the skin.
 74. The method of claim 73, whereinthe chemical is an acetone, a detergent, a depilatory agent, akeratinolytic formulation, or a combination thereof.
 75. The method ofclaim 71, wherein treating comprises applying a device.
 76. The methodof claim 75, wherein the device is selected from the group consisting ofa propellant device, a device comprising tines, a device comprisingmicroneedles, a device comprising a tine disk, a tape stripping device,a gas powered gun, a swab, an emery board, an abrasive pad, anelectroporation device, an ultrasound device, and an iontophoresisdevice.
 77. The method of claim 71, wherein the antigen is a nucleicacid encoding an antigen, a carbohydrate, a glycolipid, a glycoprotein,a lipid, a lipoprotein, phospholipid, a polypeptide, a protein, a fusionprotein, or chemical conjugate of a combination thereof.
 78. The methodof claim 71, wherein the antigen is derived from a pathogen.
 79. Themethod of claim 78, wherein the pathogen is a virus, a bacterium, aparasite, or a fungus.
 80. The method of claim 79, wherein the virus isan influenza virus or a rabies virus.
 81. The method of claim 80,wherein the antigen is hemagglutinin A.
 82. The method of claim 79,wherein the bacterium is E. coli or Bacillus anthracis.
 83. The methodof claim 82, wherein the antigen is E. coli heat-labile enterotoxin(LT).
 84. The method of claim 81, wherein the antigen is a nucleic acidencoding E. coli heat-labile enterotoxin (LT).
 85. The method of claim71, wherein the antigen is a pathogen.
 86. The method of claim 85,wherein the pathogen is a virus, a bacterium, a parasite, or a fungus.87. The method of claim 86, wherein the virus is a whole virus, a livevirus, an attenuated live virus, an inactivated virus, a detergenttreated virus, or a combination thereof.
 88. The method of claim 87,wherein the virus is an influenza virus or a rabies virus.
 89. Themethod of claim 88, wherein the influenza virus comprises hemagglutininA.
 90. The method of claim 86, wherein the bacterium is E coli orBacillus anthracis.
 91. The method of claim 90, wherein the E. colicomprises E. coli heat-labile enterotoxin (LT).
 92. The method of claim71, wherein the antigen is a multivalent antigen.
 93. The method ofclaim 71, wherein the adjuvant comprises a molecule selected from thegroup consisting of a bacterial ADP-ribosylating exotoxin (bARE), abinding B subunit of a bARE, a toxoid of a bARE, a genetically alteredbARE, and a genetically detoxified mutant of a bARE.
 94. The method ofclaim 93, wherein the antigen is an influenza antigen.
 95. The method ofclaim 71, wherein the adjuvant is a nucleic acid encoding a moleculeselected from the group consisting of a bacterial ADP-ribosylatingexotoxin (bARE), a binding B subunit of a bARE, a toxoid of a bARE, agenetically altered bARE, and a genetically detoxified mutant of a bARE.96. The method of claim 95, wherein the nucleic acid encodes E. coliheat labile enterotoxin (LT).
 97. The method of claim 71, wherein theadjuvant is selected from the group consisting of nucleic acid encodingan adjuvant, bacterial exotoxin, cytokine, chemokine,lipopolysaccharide, a molecule containing umethylated CpG motifs, a heatshock protein, a derivative of a heat shock protein, tumor necrosisfactor, genetically detoxified toxin, and combinations thereof.
 98. Themethod of claim 71, wherein the adjuvant is provided as a nucleic acidcomprising a sequence encoding the adjuvant.
 99. The method of claim 71,wherein the antigen or adjuvant activates an antigen presenting cell.100. The method of claim 99, wherein the antigen presenting cell is aLangerhans cell or a dermal dendritic cell.
 101. The method of claim 71,wherein the antigen is a whole microorganism, a whole cell, or a virion.102. The method of claim 71, wherein the first formulation isadministered subcutaneously, intradermally, or intramuscularly.
 103. Themethod of claim 71, wherein the second formulation is applied using apatch.
 104. The method of claim 7, wherein the nucleic acid is a DNAencoding an influenza protein.
 105. The method of claim 42, wherein thenucleic acid is a DNA encoding an influenza protein.
 106. The method ofclaim 77, wherein the nucleic acid is a DNA encoding an influenzaprotein.
 107. The method of claim 28, wherein the LT is a geneticallyaltered toxin.
 108. The method of claim 63, wherein the LT is agenetically altered toxin.